Guanylhydrazones and their use to treat inflammatory conditions

ABSTRACT

This invention concerns new methods and compositions that are useful in preventing and ameliorating cachexia, the clinical syndrome of poor nutritional status and bodily wasting associated with cancer and other chronic diseases. More particularly, the invention relates to aromatic guanylhydrazone (more properly termed amidinohydrazone) compositions and their use to inhibit the uptake of arginine by macrophages and/or its conversion to urea. These compositions and methods are also useful in preventing the generation of nitric oxide (NO) by cells, and so to prevent NO-mediated inflammation and other responses in persons in need of same. In another embodiment, the compounds can be used to inhibit arginine uptake in arginine-dependent tumors and infections.

This application is a continuation in part of application Ser. No.08/184,540 filed Jan. 21, 1994, now abandoned.

1. INTRODUCTION

This invention concerns new methods and compositions that are useful intreating inflammatory conditions, e.g., preventing and amelioratingcachexia, the clinical syndrome of poor nutritional status and bodilywasting associated with cancer and other chronic diseases. Moreparticularly, the invention relates to aromatic guanylhydrazone ("Ghy",more properly termed amidinohydrazone, i.e., NH₂ (CNH)--NH--N=)compositions and their use to inhibit the uptake of arginine bymacrophages and/or its conversion to urea. These compositions andmethods are also useful in preventing the generation of nitric oxide(NO) and the secretion of cytokines by macrophages and other cell types,and so to prevent NO-mediated inflammation and other responses inpersons in need of same. In another embodiment, the compounds can beused to inhibit arginine uptake in arginine-dependent tumors andinfections.

2. BACKGROUND OF THE INVENTION

Cachexia is a syndrome characterized by the wasting of tissue mass indiseased animals, and is grossly reflected as a loss of host weight.Cachexia is a progressive and often fatal complication found in manydifferent chronic disease states and its consequences require that thegoals of therapy should not be solely to redress the underlying disease.The loss of protein stores, loss of body weight and generally poornutritional status of cachectic patients can be independent sources ofmorbidity and mortality. Also, the debilitation associated with cachexiais a significant limitation on the patient's ability to tolerateaggressive medical and surgical therapies which are directed to theprimary etiology.

2.1. THE SIGNS OF CACHEXIA ARE DISTINCT FROM THOSE OF STARVATION

Cachexia is a severe, often life-threatening complication commonlyencountered in association with a variety of insults: cancer,chemotherapy, radiation injury, chronic infection, trauma and surgicalstress. Food intake insufficient to meet the total energy needs of thehost is a constant element of the cachectic syndrome. In addition tothis relative hypophagia which is a defining feature of cachexia,anorexia is also frequently encountered.

However, studies of the syndrome indicate that cachexia is not simplydue to a dietary intake of protein and carbohydrate below the needs ofthe host. Cachexia differs from unstressed caloric deprivation in thatthe pattern of wasting seen during partial or complete starvation isassociated with an initial whole body lipid loss concurrent with arelative conservation of tissue protein. By contrast, cachexia ischaracterized by the significant loss of both lipid and protein fromtissue reservoirs

2.2. THE IMPLICATIONS OF THE DIFFERENCES BETWEEN CACHEXIA AND STARVATION

The most commonly accepted general explanation for cachexia is that thehost's proteins are broken down in the tissues to provide a source ofamino acids. These amino acids in turn are thought to be needed for thesynthesis of glucose, albumin and host defense proteins in the liver.

This well-accepted theory suggests that therapies directed towardincreasing the total intake of calories and proteins shouldsubstantially ameliorate the cachectic syndrome. However, even asdrastic an intervention as total parenteral nutrition is not able toeffectively treat cachexia. (Brennan, 1986 NEJM 305:375, Detsky, et al.,1987, ANN INTERN MED 107:195, McGeer, et al., 1989, ANN INTERN MED 110:734, Koretz, 1984, J CLIN ONCOL 2:534).

In addition to the failure of supplementary nutrition as a therapeuticmodality, the fundamental difference in the pattern of the losses oflipid and protein between cachexia and starvation also indicates thatcachexia is neither the result solely of the abnormally increasednutritional needs due to the underlying disease nor of anorexia due tothe disease's disruption of the physiologic regulation of appetite.Rather the differences suggest the presence of some fundamental changesin the host's metabolism due, directly or indirectly, to the underlyingdisease.

Further supporting this conclusion has been an accumulation of evidenceimplicating soluble host-produced regulatory and effector proteins,known as cytokines, in the chain of events which leads to cachexia.Experiments have been conducted in which the blood circulation of anormal and a cachectic animal were joined. In these experiments withso-called "parabiosed" animals, it was observed that the otherwisenormal animal rapidly developed cachexia although the underlying diseaseremains entirely with the original host. These and other observationsstrongly implicate circulating mediators as the proximal cause ofcachexia, i.e., this catabolic condition is not the passive result ofthe excessive metabolic demands imposed by the growth of the invadingcells or organisms, nor the simple result of a lesser food intake thanthat required to meet metabolic demands.

One possibility as to the identity of these humoral factors was that thesoluble mediators produced in cachexia were the same molecules as hadbeen already identified as host immune/inflammatory-related molecules(cytokines), and shown to be secreted by lymphocytes and macrophages.The theory that these cytokines were involved in cachexia was confirmedby the observation that the administration of exogenous Tumor NecrosisFactor (also known as cachectin, herein abbreviated "TNF") to testanimals mimicked many features of cachexia. (Darling et al., 1990,CANCER RES 50:4008; Beutler & Cerami, 1988, ADV IMMUNOL 42:213).Further, anti-TNF antisera are able to ameliorate many, but not all, thesigns of cachexia in experimental tumor systems. (Sherry, et al., 1989,FASEB J 3:1956; Langstein, et al., 1989, SURG FORUM 15: 408).

2.3. THE IMPACT OF THE ROLE OF CYTOKINES IN CACHEXIA ON THE SEARCH FORTHERAPIES

It should be clear that even if all aspects of the cachectic syndromewere attributable to some single cytokine or, alternatively, to theactivity of some combination of several cytokines, hormones and otherhumoral factors, such knowledge would not in and of itself provide acellular or biochemical mechanism to explain the metabolic changes thatunderlie cachexia, nor lead directly to an effective therapy.Ultimately, cytokines must interact with target cells and inducemetabolic or phenotypic changes in their targets to be of physiologicaland pathophysiological significance. Thus the general identification ofcytokine mediation and the specific implications of particular cytokinemediators are only intermediate objectives in the determination ofprecisely what cellular and/or systemic metabolic changes occur to bringabout the full cachectic picture.

The findings that implicate cytokines as mediators of the variouscachectic syndromes, combined with the widespread focus on liver andperipheral muscle as major contributors to the metabolic changes ofcachexia prompted many to look at the effects of known cytokines onliver and muscle cells and to search for new cytokines having an effecton these tissues. To date, however, no known cytokines, individually orin combination, have been shown to directly mobilize amino acids fromprotein, stores in in vitro systems using cells typical of thesepresumed sites of protein breakdown in vivo.

2.4. NITRIC OXIDE AS A MEDIATOR OF ENDOTOXIC SHOCK

Nitric oxide (NO), a molecule produced enzymatically from L-arginine bynitric oxide synthase (NOS), is a mediator of both physiologicalhomeostasis and inflammatory cytotoxicity. Moncada, S. & Higgs, A.,1993, THE NEW ENGLAND JOURNAL OF MEDICINE 329, 2001-2012; Nathan, C.,1992, FASEB J 6, 3051-3064. NO production via the constitutive andinducible isoforms of NOS in endothelial cells for instance, causesvasodilation and governs blood pressure and tissue perfusion. Kilbourn,R. G., Jubran, A., Gross, S. S., et al., 1990, BIOCHEM. BIOPHYS. RES.COMMUN. 172, 1132-1138. NO production by an inducible NOS in activatedmacrophages, on the other hand, confers cytotoxic, increases vascularpermeability, and enhances the release of TNFα and IL-1. Ding, A. H.,Nathan, C. F. & Stuehr, D. J. J Immunol. 141, 2407-2412 (1988); Kubes,P. & Granger, D. N. Am. J. Physiol. 262, H611-H615 (1992); Van Dervort,A. L., Yan, L., Madara, P. J., et al. J Immunol. 152, 4102-4109 (1994);Bouskela, E. & Rubanyi, G. M. SHOCK 1, 347-353 (1994); Hibbs, J. B.,Taintor, R. R. & Vavrin, Z. Science 235, 473-476 (1987); Granger, D. L.,Hibbs, J. B., Perfect, J. R. & Durack, D. T. J. Clin. Invest. 85,264-273 (1990). Insight into the diverse biological actions of NO hasbeen facilitated by compounds that interfere with NOS to inhibitproduction of NO. Because the NOS isoforms are highly conserved,however, the available NOS inhibitors have not been found todiscriminate significantly between the activities of constitutive versusinducible NOS.

Previously available NOS inhibitors have had limited success inimproving survival from endotoxemia, in part because theyindiscriminately suppress endothelial-derived relaxing factor (EDRF),Cobb, J. P., et al., 1992, J. EXP. MED. 176: 1175-1182; Minnard, E. A.,et al., 1994, ARCH SURG. 129: 142-148; Billiar, T. R., 1990, et al., JLEUKOCYTE BIOL. 48:565-569. Suppression of EDRF during endotoxemia mayimpair survival by causing vasoconstriction and a diminution of bloodflow to critical vascular beds. Hertofore there have been reported nocompounds that inhibit cytokine-inducible macrophage NO without alsoinhibiting endothelial-derived NO.

3. SUMMARY OF THE INVENTION

The present invention relates to methods and compounds for treatingcachexia by inhibiting the production of urea, more particularly theproduction of urea by macrophages and the inhibition of the transportprocesses which mediate arginine uptake, particularly by macrophages.The method of the invention may also be used to limit or prevent thedamage induced by NO-mediated responses associated with stroke, shock,inflammation and other NO-related conditions. Another object of theinvention relates to inhibiting arginine uptake in the treatment oftumors or infections, where the tumor cells, the infected cells or theinfectious agent requires arginine. A further object of the invention isthe inhibition of the deleterious secretion of cytokines, such as TumorNecrosis Factor, by activated macrophages.

The class of compounds useful for the purposes of the invention includesbut is not limited to aromatics substituted with multipleguanylhydrazone (Ghy) moieties, more properly termed amidinohydrazones.The synthesis and use of such compounds is described. The inventionfurther encompasses screening assays to test additional compounds forthe above-noted activities, and pharmaceutical compositions useful inthe practice of this method of therapy.

Because of the close relationship between urea production and thephysiological synthesis of nitric oxide, the compounds of this inventioncan also be effective in limiting the cellular production of NO,particularly by macrophages.

While not limited to any theory of how or why the therapies describedand claimed herein operate, the invention is based, in part, on theApplicants' development of the following model for cachexia: incachexia, activated macrophages deplete the host's nitrogen pool byconverting circulating arginine to nitrogenous end products that areeliminated from the body, requiring protein catabolism by the muscleand/or liver and other organs in order to replace the lost serumarginine. Thus, activated macrophages create a "nitrogen sink" thatpersistently drains nitrogen from the systemic pool, forcing the body tocompensate by catabolizing tissue proteins to liberate amino acids asnew sources of nitrogen. The model is based on the Applicants'experiments, that, while seeking to identify a factor released byactivated macrophages which caused other tissues to make urea, foundthat activated macrophages themselves directly synthesize urea bybreaking down arginine.

Activated macrophages break down arginine in two ways: (a) into urea andornithine, or (b) into citrulline and nitric oxide (FIG. 1). The ureaand nitric oxide so generated remove nitrogen from the whole bodynitrogen pool since these metabolites cannot be efficiently recycled forreuse. Therefore, in cachexia, activated macrophages deplete the plasmaof arginine. The body's mechanisms to maintain arginine homeostasis inthe plasma will then require catabolism of protein from the muscle,liver and other organs to liberate sources of nitrogen and arginine. Thediscovery that arginine breakdown in the activated macrophage is aproximal cause of the inappropriate mobilization of tissue proteinstores allowed for the development of the therapies describedherein--i.e., interfering with arginine uptake by activated macrophages,and/or the production of urea from arginine to stem the inexorable lossof urea down the metabolic "nitrogen sink" and thus inhibit the furthercatabolism of protein from the tissues.

It is a further object of invention to provide compounds and methods oftheir use to inhibit the production of nitric oxide by macrophages toovercome the limitation imposed by the absence of a macrophage-specificinhibitor of nitric oxide synthetase. Without limitation as to theory,we reasoned that advantage could be made of the requirement for arginineuptake by macrophages but not endothelial cells induced to produce NO.The compounds of the present invention were found to inhibit theproduction of NO by macrophages while having no inhibition on theproduction of vaso-active (endothelial derived) NO. Thus, the compoundsof the invention may advantageously be used to counteractmacrophage-induced, NO-mediated effects which accompany, for example,endotoxic and septic shock.

The foregoing is presented by way of illustration and not limitation.While the invention was developed with the background knowledge of themodel for the physiology of cachexia, the invention itself involves theuse of arginine analogs, arginomimetics, and other compounds having anon-metabolizable guanylhydrazone group(s) to inhibit the macrophageproduction of urea. A general class of such compounds are aromaticscontaining guanylhydrazones. These compounds can be synthesized by thereaction of acetylbenzenes and benzaldehydes with aminoguanidine andacid at high temperature in aqueous ethanol. (Ulrich, et al., 1982, DRUGDEVELOPMENT RESEARCH 2:219; and Ulrich & Cerami, 1984 MEDICINALCHEMISTRY 27:35, which are hereby incorporated by reference.)

The invention is demonstrated by working examples describing thesynthesis of compounds used in accordance with the invention (Section 6,infra); a whole cell assay to identify compounds that inhibit ureaoutput (Section 7, infra); a whole cell assay to identify compounds thatinhibit arginine uptake (Section 8, infra); an arginase inhibition assay(Section 9, infra); the demonstration of efficacy of the invention in ananimal model system for cachexia (Section 10, infra); the demonstrationof efficacy of the invention to reduce inflammation in the animal modelusing carrageenan-induced paw swelling (Section 11, infra); thedemonstration of in vivo efficacy of the invention in preventinglipopolysaccharide-induced fatality (Section 13, infra) without blockingthe effects of endothelial-derived relaxing factor (Section 12, infra)and of in vitro efficacy of the invention in preventing the secretion ofTumor Necrosis Factor by RAW 264.7 cells stimulated by LPS andγ-interferon (Section 14, infra). The results of Sections 12-14 areindicative of efficacy in preventing the morbidity and mortalityassociated with toxic shock or sytemic inflammatory response syndrome.Further examples show the efficacy of Compound No. 14 in reducing theseverity of infarction induced by occlusion of the middle cerebralartery of a rat (Section 15, infra) and in reducing the growth of anexperimental neoplasm in a nude mouse model (Section 16, infra).

4. BRIEF DESCRIPTION OF FIGURES

FIG. 1. Biochemistry of arginine degradation by inflammatory cells andinter-organ substrate cycling.

FIG. 2. Urea production by resident macrophages.

FIG. 3. Urea production by macrophages is stimulated by LPS andγ-interferon. Control --◯--; IFNγ (25 U/ml) ----; LPS (100 ng/ml)--⋄--; IFNγ (25 U/ml)+LPS (100 ng/ml) --♦--.

FIG. 4. Dose-response relationship of the effect of γ-interferon and LPSon RAW 264.7 cell production of urea in the presence and absence ofcomplementary drugs. FIG. 4A. Various doses of γ-interferon with andwithout 100 ng/ml LPS; FIG. 4B. Various doses of LPS with and without 25U/ml γ-interferon.

FIG. 5. Dependence of RAW 264.7 urea production on extracellulararginine. Control ----; IFNγ (25 U/ml)+LPS (100 ng/ml) --♦--.

FIG. 6. Arginine transport. Uptake of tetra-³ H-arginine by RAW 264.7cells a various times after stimulation.

FIG. 7. Chemical structures of exemplary compounds of the invention.FIG. 7A. Nos. 1-7; FIG. 7B. Nos. 8-13; FIG. 7C. Nos. 14-20; FIG. 7D.Nos. 21-24; FIG. 7E. Nos. 25-27; FIG. 7F. Nos. 28-30; FIG. 7G. Nos.31-33; FIG. 7H. Nos. 34-36; FIG. 7I Nos. 37-40; FIG. 7J. Nos. 41-43.

FIG. 8. The dose dependency of the protective effects Compound No. 14 onλ-carrageenan induced mouse paw swelling.

FIG. 9. Comparison of the effect on acetylcholine-induced hypotension ofCompound No. 14 and of the known nitric oxide synthase inhibitor,L-N^(G) -methyl-arginine.

FIG. 10. Effects of Compound No. 14 on LPS-induced mortality.

FIG. 11. Effects of Compound No. 14 on the secretion of TNF byLPS/γ-interferon-stimulated RAW 264.7 cells.

FIG. 12. Western blot of medium from LPS/γ-IFN-stimulated RAW 264.7cells showing the production of TNF by cells treated with 0, 1, 5 and 25μM Compound No. 14.

FIG. 13. Comparison of the combined effects of Compound No. 14 andextracellular arginine on the production of NO and TNF byLPS/γ-IFN-stimulated RAW 264.7 cells. FIG. 13A. NO production: Control----; 1 μM No. 14 --∇--; 5 μM No. 14 --▾--; 25 μM No. 14 --□--. FIG.13B. TNF production: Control --574 --; 5 μM No. 14 --▾--; 25 μM No. 14--∇--;

FIG. 14. Effects of Compound No. 14 on the production of cytokines byLPS/γ-IFN-stimulated PBMC. FIG. 14A. Tumor Necrosis Factor; FIG. 14B.IL-6; FIG. 14C. Macrophage Inflammatory Protein-1α FIG. 14D. MacrophageInflammatory Protein-1β.

FIG. 15. Effects of Compound No. 14 on the transport of arginine inresting, ----, and stimulated RAW 264.7 cells, --∇--.

FIG. 16. Effects of Compound No. 14 on NO output of RAW 264.7 cellsstimulated by γ-IFN/LPS for 8 hours and exposed to Compound No. 14 forfurther 4 hours.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compounds for treatingcachexia by inhibiting the production of urea, more particularly theproduction of urea by macrophages. The method of the invention may alsobe used to limit or prevent the damage induced by NO-mediated responsesassociated with stroke, shock, inflammation and other NO-relatedconditions. To this end, the present invention relates to the inhibitionof macrophage production of urea and NO, and more particularly,inhibition of induced excessive production of urea and the inhibition ofthe transport processes which mediate arginine uptake by macrophages.The invention further includes the inhibition of the deleterioussecretion of cytokines, e.g. Tumor Necrosis Factor (TNF). An alternateembodiment of the invention relates to the inhibition of arginine uptakein the treatment of tumors or infections, where the tumor cells or theinfectious agent requires arginine.

The class of compounds useful for the invention includes aromaticssubstituted with multiple guanylhydrazone moieties, more properly termedamidinohydrazones. The synthesis and use of such compounds is described.The invention further encompasses screening assays to test additionalcompounds for such activity and pharmaceutical compositions useful inthe practice of this method of therapy.

Historically, our initial studies were directed to the discovery of anhypothesized macrophage-produced soluble mediator which caused culturedhepatocytes to produce more urea. More particularly, we undertook tofind a mediator produced by stimulated or activated macrophages such asare present in many chronic disease states associated with cachexia.Control studies performed during the initial attempts to isolate acytokine which caused increased hepatocyte urea production in vitro ledto the serendipitous discovery that the activated macrophages themselvesproduce urea in substantial amounts. That this was relevant to in vivoconditions was confirmed by experiments demonstrating enhanced ureaproduction by macrophages isolated from mice made cachectic by atransplanted tumor (FIG. 2). These studies were further confirmed by thedemonstration that the murine macrophage line RAW 264.7 responds to themacrophage activators LPS and γIFN with increased urea production whichdepends on the extracellular availability of arginine (FIGS. 3-5). Thesestudies led to the assay for pharmacologic activity, which is describedin detail herein below. The compounds used in the instant invention wereidentified based on this assay and used in animal model systems whichdemonstrate the efficacy of the invention (Sections 10, 11, 12, 13, 15and 16, infra).

The foregoing results indicate that the secretion of cytokines byactivated macrophages is but one aspect of the cachectic process. Wedeveloped the following model and hypothesis which is presented forexplanatory purposes only and without limitation of the invention to anyparticular mechanism or scientific model. Equally important, in thismodel, are the direct metabolic processes of activated macrophages. Thedata indicate that in cachexia, activated macrophages make and secreteurea abundantly. The magnitude of the nitrogen loss which can bedirectly attributed to macrophages can be estimated from experimentalculture data. Such studies show a single activated macrophage producesabout 50 pg of urea/day. If one estimates that the fraction of activatedmacrophages is about 10% of the whole body immune cell population in ahuman being, then there is a total of about 10¹¹ activated macrophages.This population could correspondingly account for the loss of about 5grams of nitrogen per day which translates roughly to 30 grams ofprotein per day. This magnitude of loss represents a significantfraction of the weight loss that is observed clinically over time inchronic cachexia.

In accordance with the invention, inhibition of urea production,particularly that induced in macrophages, will reverse the process.Macrophage cellular metabolism differs from hepatic metabolism regardingthe enzymes available to complete the so-called "urea cycle." In bothtissues the ultimate step in urea production is a hydrolytic cleavage bythe enzyme arginase of the amidino moiety of arginine to yield ornithineand urea (FIG. 1). One salient difference between macrophage and hepaticurea production is that macrophages lack substantial quantities of theenzyme ornithine transcarbamoylase and hence they cannot efficientlysalvage the ornithine produced by arginase nor can they directly useammonium (NH₄) to form urea but rather must rely on an exogenous supplyof arginine. Secondly, it appears that there are two arginase enzymes,both of which catalyze the hydrolytic release of urea, one foundespecially in macrophages and a second found typically in hepatocytes ofthe liver.

These differences between macrophage and hepatic urea production havetwo implications: first, macrophages will selectively deplete argininefrom the plasma. This circulating arginine must ultimately be replacedby protein breakdown in other tissues because the conversion of nitrogento urea is essentially irreversible, i.e., urea cannot be furthermetabolized for re-use. Arginine itself is synthesized from α-ketoglutarate and ammonium by glutamate synthetase, glutamine synthetase andcarbamoyl phosphate synthetase. Secondly, given that macrophage ureaproduction depends upon arginine uptake while hepatic urea synthesisdoes not, it may be possible to selectively block thecachexia-associated nitrogen loss while leaving corresponding hepaticfunctions relatively undisturbed.

In addition it may be advantageous to specifically block the macrophageform of the arginase enzyme and not the liver form. In vitro assays aredescribed herein to detect the degree to which test compoundsspecifically inhibit each of these metabolic processes. These assays usethe macrophage cell line RAW 264.7. Twenty compounds, including 15 novelcompounds were tested for inhibition of macrophage urea production in anRAW 264.7 cell line assay. Six of the compounds display an IC₅₀ of about10 μM or less and a further five compounds have been noted with an IC₅₀of greater than 10 μM but less than about 100 μM (see Section 7.2).

Alternative embodiments of the present invention encompass any knownmeans to inhibit macrophage urea production. Such methods may includebut are not limited to the use of recombinant DNA methodologies. Forexample, a vector expressing an antisense message complementary to themRNA of the macrophage form of arginase will be introduced into themacrophages of the cachectic host. Alternatively, a ribozyme specificfor the mRNA of the macrophage form of arginase could be employed.Specific introduction into macrophages of vectors appropriate to eitherwill be obtained by use of liposome carriers.

Another embodiment of the present invention involves inhibiting nitricoxide (NO) production and particularly of the enzyme NO-synthase. NO isproduced by activated macrophages and vascular endothelial cells amongother cellular sources. NO has been implicated as a causativepathological factor in a variety of inflammatory conditions:particularly in circumstances of shock and of ischemic necrosis(infarction) of the myocardium and of the central nervous system.NO-synthase catalyzes the oxidation of arginine to citrulline with anaccompanying release of NO. Compounds which inhibit arginine uptake willtherefore be effective suppressors of NO-synthase activity at thecellular level. Further, compounds of the above noted classes may bespecific inhibitors of NO-synthase at the molecular level. The datashown herein demonstrate that the compounds used in accordance with theinvention inhibit NO-production without inhibiting EDRF activity.

In a still further embodiment, the invention may be used to treat toxicshock, also known as systemic inflammatory response syndrome (SIRS). Thedata described herein shows that the compounds of the invention act bytwo independent pathways to prevent the mortality and morbidityassociated with SIRS: (a) by preventing arginine uptake and, thereby,blocking the synthesis of NO by activated macrophages; and (b) byblocking the secretion of cytokines such as Tumor Necrosis Factor (TNF).

In yet another embodiment of the invention, inhibition of arginineuptake may be used to treat tumors or infections in which the tumorcells or infectious agent requires arginine. For example, tumors witharginine requirements include but are not limited to tumors of thebreast, liver, lung and brain; whereas infectious agents with argininerequirements include but are not limited to Pneumocystis carinii,Trypanosoma brucei, T. congolense and T. evansi.

Examples of inhibitors which could be used in accordance with theinvention include, but are not limited to, analogs of arginine; moreparticularly to a class of arylene compounds substituted with[(aminoiminomethyl)hydrazono]methyl moieties and[2-(aminoiminomethyl)hydrazono]ethyl moieties (hereinafter collectively"guanylhydrazones"); most preferably diphenyl compounds having 2, 3 or 4guanylhydrazone moieties. These inhibitory compounds have one or morenon-hydrolyzable analogs of the guanidino group of arginine.

Of guanylhydrazone compounds examined for activity in the presentinvention, compounds having only a single guanylhydrazone moiety wereeither inactive or required mM concentrations to achieve a 50% reductionin urea output. Benzyl and diphenyl compounds having 2, 3 or 4guanylhydrazone moieties were active in some cases at less than 10 μM.In all cases tested the highly active compounds inhibited not only ureaproduction but also inhibited the transport of arginine into the cell. Apreferred embodiment of the present invention are di, tri and tetraguanylhydrazone substituted phenyl compounds having two phenyl nucleilinked by an alkanediamide or two phenoxy nuclei linked by an alkane. Asecond preferred embodiment are triacetylphenyl or triformylphenyltris(guanylhydrazones). Examples of such useful compounds which wereknown include monoarylene bisguanylhydrazone, e.g., 1,3-diacetylpyridinebis(guanylhydrazone) (2), Ulrich, 1982, a monoarylenetris(guanylhydrazone), e.g., 1,3,5-triacetylbenzenetris(guanylhydrazone) (1), Ulrich, 1984, and a bisarylenebis(guanylhydrazone), e.g., 4,4'-diacetyldiphenylureabis(guanylhydrazone) (8), Korytnyk, W. et al., J. MEDICINAL CHEMISTRY21:507-13, 1978. These compounds inhibit macrophage urea production invitro at concentrations of between about 10 μM and about 50 μM. Furthernovel compounds of the present invention have inhibitory activity atfive-fold lower concentrations. Such compounds include a bisarylenetris(guanylhydrazone), e.g., 3,5,4'-triacetyldiphenylureatris(guanylhydrazone) (FIG. 17.9), a bisarylene tetrakis(guanylhydrazone), e.g., N,N'-bis(3,5-diacetylphenyl)decanediamidetetrakis (guanylhydrazone) (FIG. 17.14) and3,3'-(ethylenedioxy)dibenzaldehyde bis(guanylhydrazone) (FIG. 17.16).

Further contemplated within the scope of the invention are tris aryleneguanylhydrazono compounds in which each arylene group bears 1 or 2guanylhydrazonoalkyl substituents. Such compounds may be synthesizedusing the methods taught herein.

5.1. ASSAYS FOR IDENTIFYING ACTIVE COMPOUNDS

The following assays can be used to identify compounds that are used inthe invention. Moreover, the assays can be utilized to determine theIC₅₀ (i.e., the concentration which achieves a half-maximal inhibitionof the parameter assayed) for each compound tested. When used in vivo,the dose of each compound should be formulated to achieve a range ofcirculating concentrations that include the IC₅₀ measured in vitro.

The assays are exemplary and not intended to limit the scope of themethod of the invention. Those of skill in the art will appreciate thatmodifications can be made to the assay system to develop equivalentassays that obtain the same result. In the working examples describedherein, the RAW 264.7 cell line was used. However, the invention is notlimited to the RAW 264.7 cell line which could be replaced by anymacrophage cell line or by activated non-transformed macrophages in aprimary culture.

5.1.1. WHOLE CELL ASSAY FOR UREA AND NITRIC OXIDE PRODUCTION

In general, the whole cell assay for urea production may be conducted asfollows: macrophages or endothelial cells are activated (e.g., usingfactors including but not limited to γIFN and LPS as described in theexamples, infra) in the presence of test inhibitors. After anappropriate time in culture, e.g., approximately overnight up to 2 days,the culture supernatant is analyzed for the presence of urea and nitricoxide. The production of urea and nitric oxide by any cell line can bemeasured by the same colorimetric assays used in clinical laboratoriesto determine the serum concentrations of these same compounds. Theeffects of various concentrations of inhibitor can be determined bycomparison with the supernatant of control cultures which were nottreated with the test inhibitor. A further control to indentify toxicityat any inhibitory dose may be included. An assay for the release of theintracellular enzyme lactate dehydrogenase, as used in the examplesdescribed infra, or an equivalent control, may be employed to such ends.

5.1.2. WHOLE CELL ASSAY FOR ARGININE UPTAKE

Inhibition of arginine transmembrane concentrating activity by thecompounds of the present invention is measured using carrier-freeradiolabelled arginine. To this end, the cells are cultured to allowthem to adhere (e.g., for 2 hours to overnight) and activated (e.g.,using factors including but not limited to γIFN and LPS as described inthe working examples infra) in the presence of the test inhibitor. Afteran appropriate incubation time, carrier-free labeled arginine is addedto the culture. After a short time period (e.g., 5 minutes) the cellsare washed with a solution containing unlabeled arginine to displace anyradiolabeled arginine non-specifically bound to the cells in culture.The cells are then lysed and the cell lysates analyzed for the presenceof radiolabeled arginine. The effects of the test compounds aredetermined by comparison of incorporation of radiolabel into treatedcells versus the control cell cultures which were not treated with thepotential inhibitor.

In the embodiment described in the working example herein, a testpopulation of cells was cultured for about 3 hours so that the cellsbecame firmly adherent. Various concentrations of the potentialinhibitors were then added to parallel cultures. One hour later themacrophage stimulators, e.g. including, but not limited to LPS and γIFN,were added. Eighteen hours later the cells were washed in a warmbalanced salt solution supplemented with glucose. Carrier-freeradiolabelled arginine was added; after 5 minutes, active uptake ofarginine was stopped by washing the cell three times with buffer,chilled to 0° C., containing 10 mM unlabeled arginine to displace anyexternally bound label. The contents of the washed cells are solubilizedin 100 μl of formic acid and counted by standard techniques.

5.1.3. CELL LYSATE ASSAY FOR ARGINASE ACTIVITY

The cell lysate assay for arginase activity involves exposing a celllysate to an arginase activation buffer in the presence of the testcompound. After an appropriate incubation period, arginine is added andthe enzyme activity of arginase is determined e.g., by measuring theurea concentration in the sample. Inhibitory activity of the testcompound is determined by comparing the results obtained to controlsamples which were not exposed to the test compound. As demonstrated inthe working examples, infra, direct inhibition of arginase is determinedby first preparing a low speed supernatant of a cell lysate at a proteinconcentration of between 0.5 and 4 mg/ml. The supernatant is mixed in a1:4 ratio with an activation buffer containing MnCl₂ and albumin andaliquots are incubated with various concentrations of the potentialinhibitory compound. After a 20 minute period of heat activation at 55°C., the solution is made to 0.25M arginine, then incubated at 37° C. for20 minutes. TCA is added to remove protein by centrifigation, then theurea concentration of the supernatant determined by colorimetric assaybased on diacetylmonoxime.

5.2. ACTIVE COMPOUNDS

By use of the above-noted in vitro bio-assays, compounds have beenidentified which are inhibitors of urea and nitric oxide production andof arginine uptake. The results of these experiments are summarized inSection 7.2. Of the twenty (20) compounds examined six (6) are effectiveinhibitors at concentrations between 1 and 10 μM: Compounds Nos. 1, 9,13, 14, 15, 16. A further six (6) compounds were effective atconcentrations of between 10 and 100 μM: Compounds No. 2, 8, 11, 18, and19. The compound which was identified as the most active (Compound No.14) was used in vivo in animal models of cachexia, NO-mediatedinflammation, endotoxin-induced shock, cerebral infarction andneoplasia. In each of these models Compound No. 14 proved to beeffective (Sections 10, 11, 13, 15 and 16 infra.)

5.2.1. COMPOUNDS AND THEIR SYNTHESIS

Hereinafter GhyCH--=NH₂ (CNH)--NH--N=CH-- and GhyCCH₃ =NH₂(CNH)--NH--N=CCH₃ --. The compounds of the invention include thefollowing two major genera. The first consists of compounds having theformula: ##STR1## wherein X₂ =GhyCH--, GhyCCH₃ -- or H--; X₁, X'₁ andX'₂ independently =GhyCH-- or GhyCCH₃ --;

Z=--NH(CO)NH--, --(C₆ H₄)--, --(C₅ NH₃)-- or --A--(CH₂)_(n) --A--,n=2-10, which is unsubstituted, mono- or di-C-methyl substituted, or amono or di- unsaturated derivative thereof; and

A, independently, =--NH(CO)--, --NH(CO)NH--, --NH-- or --O-- and saltsthereof. For ease of synthesis, a preferred embodiment includes thosecompounds wherein A is a single functionality. Also included within theinvention are compounds having the same formula wherein X₁ and X₂ =H;X'₁ and X'₂ independently =GhyCH-- or GhyCCH₃ --; Z=--A--(CH₂)_(n)--A--, n=3-8; and A=--NH(CO)-- or --NH(CO)NH--, and salts thereof. Alsoincluded are compounds wherein X₁ and X₂ =H; X'₁ and X'₂ independently=GhyCH-- or GhyCCH₃ -- and Z=--O--(CH₂)₂ --O--.

Further examples of genera of the invention include: The genus wherein:X₂ =GhyCH--, GhyCCH₃ -- or H--; X₁, X'₁ and X'₂ =GhyCH-- or GhyCCH--;and Z=--O--(CH₂)_(n) --O--, n=2-10 and salts thereof; and the relatedgenus wherein, when X₂ is other than H, X₂ is meta or para to X₁ andwherein X'₂ is meta or para to X'₁. A compound having the above formulawherein: X₂ =GhyCH, GhyCCH₃ or H; X₁, X'₁ and X'₂, =GhyCH-- or GhyCCH--;and Z=--NH--(C═O)--NH-- and salts thereof; and the related genuswherein, when X₂ is other than H, X₂ is meta or para to X₁ and whereinX'₂ is meta or para to X'₁.

Also included are compounds having the formula: ##STR2## wherein: n=3-8;X₂ and X'₂ =GhyCH--, GhyCCH₃ -- or H--; X₁ and X'₁ =GhyCH-- or GhyCCH₃--; and salts thereof; and the related genus wherein, when X₂ or X'₂ orboth are other than H, then X₂ or X'₂ are meta or para to X₁ or X'₁,respectively. Also included are compounds having the formula: ##STR3##wherein: X₂ and X'₂ =GhyCH--, GhyCCH--, or H--; X₁ and X'₁ =GhyCH-- orGhyCCH₃ --; and n=2-10 and salts thereof and the related genus wherein,when X₂ or X'₂ or both are other than H, then X₂ or X'₂ are meta or parato X₁ or X'₁, respectively.

The second major genus consists of compounds of the formula: ##STR4##wherein, X₁, X₂ and X₃, independently =GhyCH-- or GhyCCH₃ --; X'₁, X'₂and X'₃, independently =H, GhyCH-- or GhyCCH₃ --; Z=(C₆ H₃), when m₁,m₂, m₃ =0 or Z═N, when, independently, m₁, m₂, m₃ =2-6; andA=--NH(CO)--, --NH(CO)NH--, --NH-- or --O-- and salts thereof. Furtherexamples of genera of the invention include the genus wherein when anyof X'₁, X'₂ ; and X'₃ are other than H, then the correspondingsubstituent of the group consisting of X₁, X₂ and X₃ is meta or para toX'₁, X'₂ and X'₃, respectively; the genus wherein, m₁, m₂, m₃ =0 andA=--NH(CO)--; and the genus wherein m₁, m₂, m₃ =2-6 and A=--NH(CO)NH--.

The compounds of the present invention can be synthesized by means oftwo fundamental reactions. Those skilled in the art will recognize thatnumerous variants may be synthesized by means of these reactions andthat these variants have properties in common with the compounds hereindisclosed.

Reaction 1 consists of the reaction of a substituted aromatic having aprimary or secondary amine, e.g., 3,5-diacetylaniline, and a dioyldichloride, e.g., glutaryl dichloride, to yield the correspondingN,N'-diphenylalkanediamide. "Reversed" diamides can also be prepared.Acetyl and diacetylbenzoic acid can be prepared by the reaction of thecorresponding substituted toluenes and KMnO₄. The acids may be thenactivated by standard techniques and reacted with the appropriateα,ω-alkanediamines to yield the reverse "diamides". Mixed forward andreversed diamides can be synthesized by methods well known in the fieldof peptide synthesis. Thus, an N-t-butyloxycarbonyl amino acid may bereacted with a substituted aniline, followed by deprotection andreaction of the amino group with an activated substituted benzoic acid.When used herein the symbol "--NH(CO)--", unless otherwise indicated,includes the --(CO)NH-- isomer.

The method is not limited to dioyl dichlorides. The trichloridederivatives of trioyl compounds may be used to synthesize triphenylalkanetriamides in a similar fashion. Suitable triacids include cyclicacids, e.g., 1,3,5-cyclohexanetricarboxylic acid (Aldrich Chem. Co.),1,3,5-trimethyl,1,3,5-cyclohexanetricarboxylic acid (Kemp's triacid,Kemp and Petrakis, 1981, J.Org.Chem 46:5140), 1,3,5-benzinetricarboxylicacid (Aldrich Chem. Co.) and linear tricarboxylic acids such as1,2,3-propanetricarboxylic acid (Sigma Chem. Co.). The identicalreaction may be performed wherein the dioyl chloride is replaced bytrichloromethyl chloroformate to yield a diphenylurea condensationproduct. An alternative to Reaction 1 can be performed to yield a1,n-(n-alkanedioxy) diarylene by reacting the 1,n-dibromoalkane, e.g.,1,2 dibromoethane and a monohydroxylarylene, e.g.,3-hydroxyacetophenone.

Further embodiments of the invention include the use of triamines of theform H₂ N--(CH₂)_(n) --NH--(CH₂)_(q) --NH₂ wherein (n,q=2-6) and of theform Y--((CH₂)_(n) --NH₂)₃ wherein Y may be one of N (n =2-6), C(NO₂)(n=3), a C-alkane (n=1), 1,3,5-adamantanetriyl (n=3) or1,3,5-benzinetriyl (n=1-3).

In two further embodiments of the invention, an acetyl- or diacetylarylisocyanate is reacted with an alkanediamine or, alternatively, anacetyl- or diacetylaryl amine is reacted with an alkanediyl diisocyanateto yield bis-ureido intermediates which may be reacted withaminoguanidine to form the guanylhdrazono end products. The requisiteisocyanates are either commercially available or may be synthesized fromfrom the corresponding amines by reaction with phosgene, trichloromethylchloroformate, or bis(trichloromethyl) carbonate in toluene or xylene atelevated temperature.

Reaction 2 consists of the reaction of an acetophenone or benzaldehydetype moiety and an aminoguanidine to yield the condensation productwherein an imino-bonded (N═C) aminoguanidine replaces the ketone orcarbonyl moiety of the arylene thus forming a guanylhydrazone andaccompanied by the release of a water molecule.

5.2.2. PHARMACEUTICAL FORMULATIONS

Because of their pharmacological properties, the compounds of thepresent invention can be used especially as agents to treat patientssuffering from cachexia, deleterious NO-mediated responses, infarction,tumors or infections that require arginine. Such a compound can beadministered to a patient either by itself, or in pharmaceuticalcompositions where it is mixed with suitable carriers or excipient(s).

Use of pharmaceutically acceptable carriers to formulate the compoundsherein disclosed for the practice of the invention into dosages suitablefor systemic administration is within the scope of the invention. Withproper choice of carrier and suitable manufacturing practice, thecompositions of the present invention, in particular, those formulatedas solutions, may be administered parenterally, such as by intravenousinjection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levitating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

5.3. USES OF THE COMPOUNDS

For any compound used in the method of the invention, the appropriatedose is one which achieves a circulating range of concentrations whichencompass the IC₅₀ determined to be effective for that compound asreported herein in Tables I, II and III or determined in the mannerherein described. For example, when using Compound Nos. 1, 9, 13 or 15,for treating cachexia, inflammation, endotoxic shock or septic shock,infarction or neoplasm regardless of the formulation chosen, the amountadministered should be sufficient to achieve a serum concentration or acirculating plasma concentration of between about 5 μM and 100 μM. Whenusing Compound No. 14, for treating cachexia, inflammation, endotoxicshock or septic shock, infarction or neoplasm regardless of theformulation chosen, the amount administered should be sufficient toachieve a serum concentration or a circulating plasma concentration ofbetween about 0.5 μM and 10 μM. As shown in the working examples, adaily parenteral dose of Compound No. 14 of about 0.4 mg/Kg, used totreat cachexia, and a single parenteral dose of 1.0 mg/Kg, to treatLPS-induced toxicity, are effective in murine models. Those skilled inthe art will appreciate that the dose appropriate to a given route ofadministration can be determined by the application pharmacologicalmethods that are well known to those skilled in the art.

When the compounds of the present invention are used to treat chronicinflammation a dose regime should be determined by application ofstandard pharmacologic techniques using the above-noted dose ranges asinitial points. To treat acute inflammatory conditions, a single largerdose may be administered in an alternative embodiment. As shown in theworking examples, a single parenteral dose of 5.0 mg/Kg of Compound No.14 was found effective to treat such an acute event.

6. EXAMPLE: SYNTHESIS OF THE ACTIVE COMPOUNDS

This section describes in detail the synthesis and purification ofuseful intermediates and of exemplary compounds of the presentinvention.

6.1. SYNTHESIS OF INTERMEDIATE PRODUCTS

In the following it is understood that amidinohydrazone andguanylhydrazone and (aminoiminomethyl)hydrazono are synonyms.

The following reactions are used to link substituted arylene compoundsby means of alkane chains of various lengths. The bond may be an amide,a phenoxyalkane or a urea.

6.1.1. N,N'-BIS(3,5-DIACETYLPHENYL)PENTANEDIAMIDE

3,5-Diacetylaniline (531 mg) in dichloromethane (7 mL) containingpyridine (0.4 mL) was treated with 0.141 mL glutaryl dichloride. After 1hr, filtration and washing with water gave 555 mg ofN,N'-bis(3,5-diacetylphenyl)pentanediamide, mp 246°-7° C.

Analogously, the following were prepared from 3,5-diacetylaniline andthe corresponding dioyl dichlorides: N,N'-bis(3,5-diacetylphenyl)butanediamide, mp 293°-6° C.; N,N'-bis(3,5-diacetylphenyl)hexanediamide,mp 269°-70° C.; N,N'-bis(3,5-diacetylphenyl)heptanediamide, mp 200°-3°C.; N,N'-bis(3,5-diacetylphenyl)octanediamide, mp 183°-4° C.;N,N'-bis(3,5-diacetylphenyl)nonanediamide, mp 179°-80° C.;N,N'-bis(3,5-diacetylphenyl)decanediamide, mp 196°-9° C.;N,N'-bis(3,5-diacetylphenyl)dodecanediamide, mp 178°-9° C.;N,N'-bis(3,5-diacetylphenyl) (isophthalic acid diamide), mp 283°-4° C.Also analogously, N,N'-bis(3-acetylphenyl)pentanediamide, mp 174°-5° C.was prepared from 3-aminoacetophenone and glutaryl dichloride.

6.1.2. N-(4-ACETYLPHENYL)N'-(3,5-DIACETYLPHENYL)UREA

4-Aminoacetophenone (1.35 g) in toluene (20 mL) was treated withtrichloromethyl chloroformate (1.2 mL). The mixture was heated at refluxfor 2 hr. 3,5-diacetylaniline (1.77 g) was added and the mixture washeated at reflux for 1 hr then allowed to stand 16 hr at room temp. Theproduct was filtered off and washed with ethanol and dried to give 0.93g of N-(4-acetylphenyl)-N'-(3,5-diacetylphenyl)urea, mp 251°-2° C.

6.1.3. 1,2-BIS(3-ACETYLPHENOXY)ETHANE

3-Hydroxyacetophenone (8.4 g) and 1,2-dibromoethane (5.07 g) weretreated with potassium hydroxide (3.83 g) and heated under nitrogen atreflux for 2 days. The mixture was cooled and water (200 mL) was addedand the mixture stirred for 1 hr. The precipitate was filtered out andrecrystallized from isopropanol to give 1.21 g of1,2-bis(3-acetylphenoxy)ethane, mp 120°-1° C.

6.1.4.1,5-BIS[([(3,5-DIACETYLPHENYL)AMINO]CARBONY)LAMINO]-2-METHYLPENTANE

1,5-Diisocyanato-2-methylpentane (0.18 ml) was added to a suspension of3,5-diacetylaniline (0.531 g) in dichloromethane (7 ml) containingcatalytic 4-dimethylaminopyridine (10 mg). The mixture was refluxed for2 hr and allowed to stand overnight. Filtration gave while crystals,0.30 g, mp 124°-130° C.

6.1.5. TRIS[2-([(3-ACETYLPHENYL)AMINO]CARBONYLAMINO)ETHYL]AMINE

3-Acetylphenyl isocyanatate (0.60 g) in dichloromethane (10 mL) wasreacted with tris(2-aminoethyl)amine (0.146 g). A vigorous reactionoccurred and a copious white turbidity resulted. Methanol was added andthe mixture was reconcentrated to produce crystalline material which wasfiltered out. Yield 0.61 g, mp 193° C.

6.1.6. 3,5-DIACETYLPHENYL ISOCYANATE ANDN,N'-BIS(3,5-DIACETYLPHENYL)UREA

3,5-Diacetylaniline (3.0 g, 16.9 mmol) was suspended in toluene (50 mL)with stirring in an ice bath. A solution of bis(trichlormethyl)carbonate (1.67 g, 5.9 mmol) in toluene (10 mL) was added. Thesuspension was allowed to warm to room temp. and was stirred overnightat r.t. The mixture was then heated at reflux for 4 hr, cooled, andfiltered to give 1.1 g of N,N'-bis(3,5-diacetylphenyl)urea, mp dec137°-8° C. (gas evol.). The filtrate was evaporated to give 2.2 g of3,5-diacetylphenyl isocyanate as a white powder, mp 71° C.

6.2. CONVERSION OF INTERMEDIATES TO END PRODUCTS

The following reactions are examples which illustrate a generalcondensation reaction wherein the primary amine of aminoguanidinedisplaces the oxygen of an acetophenone or benzaldehyde or ketone andelaborates an H₂ O and forms the guanylhydrazone. In general, allreactions are carried out at elevated temperature with acid catalysis inaqueous alcohol. The products are recovered by crystallization uponcooling and, optionally, the addition of petroleum ether or isopropanol.Purification was performed by recrystallization.

Compound 4, FIG. 7A.4: 4-([(aminoiminomethyl) hydrazono]methyl)cinnamicacid hydrochloride:

4-formylcinnamic acid (1.76 g) and aminoguanidine hydrochloride (1.22 g)were heated in 83% ethanol (24 mL) for 2 hr. Cooling and filtration gave2.56 g of 4-([(aminoiminomethyl)hydrazono]methyl)cinnamic acidhydrochloride, mp 285°-8° C.

Compound 6, FIG. 7A.6:2-([(1H-imidazol-1-yl)-1,4-phenylene]ethylidyne)hydrazinecarboximidamidehydrochloride:

4-(1H-imidazol-1-yl)acetophenone (1.86 g) and aminoguanidinehydrochloride (1.22 g) were heated in 83% ethanol (12 mL) for 48 hr.Cooling and filtration gave 2.6 g of2-([(1H-imidazol-1-yl)-1,4-phenylene]ethylidyne)hydrazinecarboximidamidehydrochloride, mp 275°-6° C.

Compound 7, FIG. 7A.7:2-[(3,4-dihydroxyphenyl)ethylidyne]hydrazinecarboximidamidehydrochloride:

3,4-dihydroxyacetophenone (3.04 g) and aminoguanidine hydrochloride(2.44 g) were heated in 75% ethanol (16 mL) for 4 hr under nitrogen.Cooling and filtration gave 2.7 g of2-[(3,4-dihydroxyphenyl)ethylidyne]hydrazinecarboximidamidehydrochloride, mp 242°-5° C.

Compound 9, FIG. 7B.9: N-(4-acetylphenyl)-N'-(3,5-diacetylphenyl)ureatris(amidinohydrazone) trihydrochloride:

N-(4-acetylphenyl)-N'-(3,5-diacetylphenyl)urea (0.676 g), aminoguanidinehydrochloride (0.83 g), and aminoguanidine dihydrochloride (0.01 g) wereheated in 83% methanol (12 mL) for 18 hr. Cooling and filtration gave0.85 g of N-(4-acetylphenyl)-N'-(3,5-diacetylphenyl)ureatris(amidinohydrazone) trihydrochloride, mp 247°-253° C. dec.

Compound 10, FIG. 7B.10:5-(1-[2-(aminoiminomethyl)hydrazono]ethyl)salicylic acid hydrochloride:

5-Acetylsalicylic acid (3.6 g) and aminoguanidine hydrochloride (2.4 g)were heated in 80% ethanol (25 mL) for 2 hr. Cooling and filtration gave5.2 g of crude 5-(1-[2-(aminoiminomethyl)hydrazono]ethyl) salicylic acidhydrochloride. Of this, 0.58 g was purified by dissolving in aq. NaOH(pH 12.5) and reprecipitation with aq HCl (to pH 2) to give 0.45 g of5-(1-[2-(aminoiminomethyl)hydrazono]ethyl)salicylic acid hydrochloride,mp 312°-3° C. (dec).

Compound 12, FIG. 7B.12: N,N'-bis(3-acetylphenyl)pentanediamidebis(amidinohydrazone) dihydrochloride:

N,N'-bis(3-acetylphenyl)pentanediamide (3.66 g), aminoguanidinehydrochloride (2.75 g), and aminoguanidine dihydrochloride (0.05 g) wereheated in methanol (35 mL) for 18 hr. Cooling and filtration gave 5.412g of N,N'-bis(3-acetylphenyl)pentanediamide bis(amidinohydrazone)dihydrochloride, mp 187°-191° C.

Compound 13, FIG. 7B.13: N,N'-bis(3,5-diacetylphenyl)pentanediamidetetrakis(amidinohydrazone) tetrahydrochloride:

N,N'-bis(3,5-diacetylphenyl)pentanediamide (0.45 g), aminoguanidinehydrochloride (0.55 g), and aminoguanidine dihydrochloride (0.01 g) wereheated in 91% ethanol (4.4 mL) for 18 hr. Cooling and filtration gave0.794 g of N,N'-bis(3,5-diacetylphenyl)pentanediamidetetrakis(amidinohydrazone) tetrahydrochloride, mp 299°-301° C. dec.

Compound 14, FIG. 7C.14: N,N'-bis(3,5-diacetylphenyl)decanediamidetetrakis(amidinohydrazone) tetrahydrochloride:

N,N'-bis(3,5-diacetylphenyl)decanediamide (0.65 g), aminoguanidinehydrochloride (0.691 g), and aminoguanidine dihydrochloride (0.01 g)were heated in 91% ethanol (5.5 mL) for 18 hr. Cooling and filtrationgave 0.87 g of N,N'-bis(3,5-diacetylphenyl)decanediamide tetrakis(amidinohydrazone) tetrahydrochloride, mp 323°-4° C. dec.

Compound 15, FIG. 7C.15:2,2'-[1,2-ethanediylbis(oxy-3,1-phenyleneethylidyne)]bishydrazinecarboximidamide dihydrochloride:

1,2-bis(3-acetylphenoxy) ethane (0.894 g), aminoguanidine hydrochloride(0.83 g), and aminoguanidine dihydrochloride (0.01 g) were heated in aq.96% methanol (6.25 mL) for 18 hr. Cooling and filtration gave 1.378 g of2,2'-[1,2-ethane-diylbis(oxy-3,1-phenyleneethylidyne)]bis(hydrazinecarboximidamide)dihydrochloride, mp 303°-7° C.

Compound 16, FIG. 7C.16: 3,3'-(ethylenedioxy)dibenzaldehydebis(amidinohydrazone) dihydrochloride:

3,3'-(ethylenedioxy)dibenzaldehyde (1.08 g), aminoguanidinehydrochloride (1.105 g), and aminoguanidine dihydrochloride (0.005 g)were heated in 96% ethanol (6.25 mL) for 18 hr under nitrogen. Coolingand filtration gave 1.428 g of 3,3'-(ethylenedioxy)dibenzaldehydebis(amidinohydrazone) dihydrochloride, mp 264°-6° C.

Compound 17, FIG. 7C.17: 4,4'-(ethylenedioxy) di-m-anisaldehydebis(amidinohydrazone) dihydrochloride:

4,4'-(ethylenedioxy)di-m-anisaldehyde (0.99 g), aminoguanidinehydrochloride (0.829 g), and aminoguanidine dihydrochloride (0.01 g)were heated in 95% methanol (10.5 mL) under nitrogen for 16 hr. Coolingand filtration gave 1.52 g of 4,4'-(ethylenedioxy)di-m-anisaldehydebis(amidinohydrazone) dihydrochloride, mp 322°-3° C. dec.

Compound 18, FIG. 7C.18: 3,3'-(trimethylenedioxy)di-p-anisaldehydebis(amidinohydrazone) dihydrochloride:

3,3'-(trimethylenedioxy)di-p-anisaldehyde (1.032 g), aminoguanidinehydrochloride (0.829 g), and aminoguanidine dihydrochloride (0.02 g)were heated in 95% methanol (10.5 mL) for 16 hr. Cooling and filtrationgave 1.372 g of 3,3'-(trimethylenedioxy)di-p-anisaldehydebis(amidinohydrazone) dihydrochloride, mp 233°-5° C.

Compound 19, FIG. 7C.19:1,4-bis[2-(aminoiminomethyl)hydrazono]cyclohexane dihydrochloride:

1,4-cyclohexanedione (2.24 g), aminoguanidine bicarbonate (6.0 g), andconcentrated hydrochloric acid (3.67 mL) were heated in water (50 mL)for 5 min. The solution was cooled and treated with isopropanol (50 mL).After crystallization was complete, filtration gave 2.91 g of1,4-bis[2-(aminoiminomethyl)hydrazono]cyclohexane dihydrochloride, mp260° C. dec.

Compound 20, FIG. 7C.20:2,2'-(1,4-diphenyl-1,4-butanediylidene)bishydrazinecarboximidamidedihydrochloride:

1,2-dibenzoylethane (4.76 g), aminoguanidine bicarbonate (5.45 g), andconcentrated hydrochloric acid (3.33 mL) were heated in 50% ethanol (60mL) for 24 hr. Cooling, concentration and filtration gave 4.3 g of2,2'-(1,4-diphenyl-1,4-butanediylidene)bis(hydrazinecarboximidamide)dihydrochloride, mp 285°-6° C.

6.3. FURTHER EXEMPLARY COMPOUNDS

Compound 21, FIG. 7D.21: N,N'-bis(3,5-diacetylphenyl)butanediamidetetrakis(amidinohydrazone) tetrahydrochloride:

N,N'-bis(3,5-diacetylphenyl)butanediamide (0.545 g), aminoguanidinehydrochloride (0.69 g), and aminoguanidine dihydrochloride (0.01 g) wereheated in 91% ethanol (5.5 mL) for 18 hr. Cooling and filtration gave0.97 g of N,N'-bis(3,5-diacetylphenyl)butanediamide tetrakis(amidinohydrazone) tetrahydrochloride, mp 314° C.

Compound 22, FIG. 7D.22: N,N'-bis(3,5-diacetylphenyl) hexanediamidetetrakis(amidinohydrazone) tetrahydrochloride:

N,N'-bis(3,5-diacetylphenyl) hexanediamide (0.58 g), aminoguanidinehydrochloride (0.69 g), and aminoguanidine dihydrochloride (0.01 g) wereheated in 91% 2-mothoxyethanol (5.5 mL) for 18 hr. Filtration while hotgave 0.936 g of N,N'-bis (3,5-diacetylphenyl) hexanediamidetetrakis(amidinohydrazone) tetrahydrochloride, mp (chars) 320°-330° C.

Compound 23, FIG. 7D.23: N,N'-bis(3,5-diacetylphenyl)heptanediamidetetrakis(amidinohydrazone) tetrahydrochloride:

N,N'-bis(3,5-diacetylphenyl) heptanediamide (0.478 g), aminoguanidinehydrochloride (0.553 g), and aminoguanidine dihydrochloride (0.01 g)were heated in 91% ethanol (4.4 mL) for 18 hr. Cooling and filtrationgave 0.739 g of N,N'-bis(3,5-diacetylphenyl)heptanediamidetetrakis(amidinohydrazone) tetrahydrochloride, mp 273°-7° C.

Compound 24, FIG. 7D.24: N,N'-bis(3,5-diacetylphenyl) (isophthalic aciddiamide) tetrakis(amidinohydrazone) tetrahydrochloride:

N,N'-bis(3,5-diacetylphenyl) (isophthalic acid diamide) (0.726 g) andaminoguanidine hydrochloride (0.829 g) were heated in 7:12-methoxyethanol/water (11.5 mL) for 18 hr. Cooling and filtration gave0.54 g of N,N'-bis(3,5-diacetylphenyl) (isophthalic acid diamide)tetrakis(amidinohydrazone) tetrahydrochloride, (chars) mp 322°-330° C.

Compound 25, FIG. 7E.25: 3,3'-(pentamethylenedioxy)di-p-anisaldehyde(0.748 g), aminoguanidine hydrochloride (0.553 g), and aminoguanidinedihydrochloride (0.01 g) were heated in methanol (5 mL) for 18 hr.Cooling and filtration gave 0.080 g of3,3'-(pentamethylenedioxy)di-p-anisaldehyde bis(amidinohydrazone)dihydrochloride, mp 195°-8° C.

Compound 26, FIG. 7E.26: A solution of 3,5-diacetylaniline (0.885 g) intetrahydrofuran (10 mL) containing 0.45 mL pyridine was treated with0.65 mL benzoyl chloride. The mixture was stirred 1 hr, then treatedwith 1 mL water and stirred 15 min. The mixture was then diluted with 40mL water and stirred 30 min. Filtration and washing with water andisopropanol gave colorless needles of N-benzoyl-3,5-diacetylaniline,1.36 g, mp 188°-189° C. A suspension of N-benzoyl-3,5-diacetylaniline(0.844 g) in aq. 87.5% ethanol (8 mL) containing 0.73 g aminoguanidinehydrochloride and a trace of HCl was heated at reflux for 18 hr. Coolingand filtration gave 1.357 g of N-benzoyl-3,5-diacetylanilinebis(amidinohydrazone) dihydrochloride (Compound 26), mp 268°-72° C.

Compound 27, FIG. 7E.27: A suspension of 3,5-diacetylaniline (0.531 g)in water (8 mL) was treated with cyanamide (0.143 g) and conc. HCl (0.25mL) and heated at reflux. Additional 0.080 g portions of cyanamide wereadded at 2 hr and 4 hr. After 6 hr, the mixture was concentrated invacuo until crystalline material separated, then filtered to give 0.110g of off-white solid, mp 120°-2° C. Of this, 0.104 g was treated withaminoguanidine hydrochloride (0.112 g) in 2.5 mL of aq. 80% ethanolcontaining aminoguanidine dihydrochloride (0.01 g). After 18 hr atreflux, colling and filtration gave 133 mg of(3,5-diacetylphenyl)guanidine bis(amidinohydrazone) trihydrochloride(Compound 27) as a slightly off-white solid, mp 270°-3° C.

Compound 28, FIG. 7F.28: A suspension of 3,5-diacetylaniline (0.531 g)in water (8 mL) was treated with cyanoguanidine (0.285 g) and conc. HCl(0.25 mL) and heated at reflux. After 6 hr the mixture was cooled andconcentrated and 0.248 g of off-white solid was filtered out, mp 260-70(dec). Of this, 0.238 g was heated at reflux with aminoguanidinehydrochloride (0.221 g) in 5.5 mL of aq. 91% methanol for 24 hr.Filtration gave 0.290 g of N-(3,5-diacetylphenyl)biguanidebis(amidinohydrazone) trihydrochloride (Compound 28) as fine whiteneedles, mp 294°-7° C.

Compound 31, FIG. 7G.31: 4-acetylphenyl isocyanate (1.2 g) andtris(2-aminoethyl)amine (0.300 mL) in methylene chloride (10 mL) werestirred at r.t. for 30 min. Filtration gave 1.35 g oftris(2-[([(4-acetylphenyl)amino]carbonyl)amino]ethyl)amine, mp 189°-90°C. This triketone (1.0 g) and aminoguanidine dihydrochloride (0.77 g)were heated in methanol (5 mL) for 4 hr. Addition of ethanol (5 ml) andfiltration gave 1.08 g oftris(2-[([(4-acetylphenyl)amino]carbonyl)amino]ethyl)aminetris(amidinohydrazone) trihydrochloride (Compound 31) as the dihydrate,mp 224°-5° C. (dec.).

Compound 32, FIG. 7G.32: 3'-aminoacetophenone (0.446 g) and1,3,5-benzenetricarbonyltrichloride (0.266 g) in tetrahydrofuran (5 mL)were stirred at r.t. for 30 min. Filtration gave 0.500 g ofN,N',N"-tris(3-acetylphenyl)-1,3,5-benzenetricarboxamide, mp 270° C. Thetriketone (1.0 g) and aminoguanidine dihydrochloride (0.87 g) wereheated in 2-methoxyethanol (5 mL) for 4 hr. Cooling and filtration gave1.4 g of N,N',N"-tris(3-acetylphenyl)-1,3,5-benzenetricarboxamidetris(amidinohydrazone) trihydrochloride (Compound 32) solvated with onemolecule of 2-methoxyethanol, mp 270°-5° C. dec.

Compound 33, FIG. 7G.33, was prepared analogously from4'-aminoacetophenone via the triketoneN,N',N"-tris(4-acetylphenyl)-1,3,5-benzenetricarboxamide, mp 310° C., togive N,N',N"-tris(4-acetylphenyl)-1,3,5-benzenetricarboxamidetris(amidinohydrazone) trihydrochloride (Compound 33) solvated withthree molecules of 2-methoxyethanol, mp 295°-300° C. (dec) (slowheating).

Compound 34, FIG. 7H.34: 3,5-diacetylphenyl isocyanate (0.6 g) andtris(2-aminoethyl)amine (0.13 g) in methylene chloride (5 mL) werestirred at r.t. for 30 min. Filtration gave 0.6 g oftris(2-[([(3,5-diacetylphenyl)amino]carbonyl)amino]ethyl)amine, mp197°-8° C. This hexa-ketone (0.47 g) and aminoguanidine dihydrochloride(0.61 g) were heated in methanol (5 mL) for 4 hr. Cooling and filtrationgave 0.86 g of tris(2-[([(3,5-diacetylphenyl)amino]carbonyl)amino]ethyl)aminehexakis(amidinohydrazone) heptahydrochloride (Compound 34) as thedihydrate hemi-ethanolate, mp 245°-6° C. dec.

Compound 35, FIG. 7H.35: 4-acetylphenyl isocyanate (3 g) andtris(3-aminopropyl)amine (1.06 g) in tetrahydrofuran (50 mL) werestirred for 30 min. Ethanol (100 mL) was added and the mixture was leftat r.t. for overnight. Filtration gave 2.8 g oftris(3-[([(4-acetylphenyl)amino]carbonyl)amino]propyl)amine, mp 184°-5°C. This diketone (0.4 g) and aminoguanidine dihydrochloride (0.29 g)were heated in methanol (3 mL) for 4 hours. Addition of ethanol (3 mL)and filtration gave 0.5 g of tris(3-[([(4-acetylphenyl)amino]carbonyl)amino]propyl)aminetris(amidinohydrazone) trihydrochloride (Compound 35) as the dihydrateethanolate, mp 209°-10° C. dec.

Compound 36, FIG. 7H.36: 3,5-Diacetylphenyl isocyanate (0.4 g) andtris(3-aminopropyl)amine (0.12 g) in tetrahydrofuran (5 mL) were stirredat r.t. for 30 min. Filtration gave 0.32 g oftris(3-[([(3,5-diacetylphenyl)amino]carbonyl)amino]propyl)amine, mp147°-8° C. This hexaketone (0.4 g) and aminoguanidine dihydrochloride(0.49 g) were heated in methanol (3 mL) for 4 hours. Addition of ethanoland filtration gave 0.58 g oftris(3-[([(3,5-diacetylphenyl)amino]carbonyl)amino]propyl)aminehexakis(amidinohydrazone) heptahydrochloride (Compound 36) as thedihydrate hemi-ethanolate, mp 250°-1° C. (dec).

Compound 37, FIG. 71.37: 3-Acetylphenyl isocyanate (1 g) and4'-aminoacetophenone (0.84 g) in methylene chloride (5 mL) were stirredat r.t. for 30 min. Filtration gave 2.0 g of3,4'-diacetyl-N,N'-diphenylurea, mp 252°-3° C. (dec). This diketone (0.6g) and aminoguanidine dihydrochloride (0.65 g) were heated in ethanol (5mL). Cooling and filtration gave 0.8 g of3,4'-diacetyl-N,N'-diphenylurea bis(amidinohydrazone) dihydrochloride(Compound 37) as the hemihydrate, mp 243°-4° C. dec.

Compound 38, FIG. 71.38: 3-Acetylphenyl isocyanate (0.27 g) and3,5-diacetylaniline (0.3 g) were stirred in methylene chloride (5 mL) atr.t. for 30 min. Filtration gave 0.5 g of3,3',5'-triacetyl-N,N'-diphenylurea, mp 223°-4° C. This triketone (0.34g) and aminoguanidine dihydrochloride (0.49 g) were heated in ethanol (5mL) for 4 hr. Cooling and filtration gave 0.6 g of3,3',5'-triacetyl-N,N'-diphenylurea tris(amidinohydrazone)trihydrochloride (Compound 38) as the hydrate, mp 245° C. dec.

Compound 39, FIG. 71.39: N,N'-Bis(3,5-diacetylphenyl)urea (0.38 g,aminoguanidine hydrochloride (0.44 g) and aminoguanidine dihydrochloride(59 mg) were heated at 90°-100° C. in 2-methoxyethanol (5 mL) for 6hours. Cooling and filtration gave 0.66 g ofN,N'-bis(3,5-diacetylphenyl)urea tetrakis(amidinohydrazone)tetrahydrochloride (Compound 39) as the 2.5-hydrate hemi-methanolate, mp263°-4° C. dec.

Compound 40, FIG. 7I.40: N,N'-bis(3,5-diacetylphenyl)nonanediamide(0.100 g), aminoguanidine hydrochloride (0.115 g), and aminoguanidinedihydrochloride (3 mg) were heated at reflux in 95% ethanol (2.5 mL) for5 hr. Cooling and filtration gave 0.18 g ofN,N'-bis(3,5-diacetylphenyl)nonanediamide tetrakis(amidinohydrazone)tetrahydrochloride (Compound 40) as the dihydrate, mp 295°-6° C.

Compound 41, FIG. 7J.41: N,N'-bis(3,5-diacetylphenyl)dodecanediamide(0.100 g), aminoguanidine hydrochloride (0.115 g), and aminoguanidinedihydrochloride (3 mg) were heated in 95% ethanol (2.5 mL) for hr.Cooling and filtration gave 0.070 g ofN,N'-bis(3,5-diacetylphenyl)dodecanediamide tetrakis(amidinohydrazone)tetrahydrochloride (Compound 41) as the tetrahydrate, mp 268°-9° C.

Compound 42, FIG. 7J.42: A suspension of 3,5-diacetylaniline (0.354 g)in methylene chloride (7 mL) containing 4-dimethylaminopyridine (5 mg)was treated with 2-methyl-1,5-pentanediyl diisocyanate (0.18 mL). Afterheating at reflux for 2 hr, the mixture was cooled. An aliquot wastreated with t-butyl methyl ether to give seed crystals which were addedto the reaction mixture. After stirring several hr, filtration gave0.120 g of1,5-bis[([(3,5-diacetylphenyl)amino]carbonyl)amino]-2-methylpentane, mp128° C. This tetraketone (0.100 g), aminoguanidine hydrochloride (0.115g), and aminoguanidine dihydrochloride (3 mg) were heated in 95% ethanol(2.5 mL) for 18 hr. Cooling and filtration gave 0.16 g of1,5-bis[([(3,5-diacetylphenyl)amino]carbonyl)amino]-2-methylpentanetetrakis(amidinohydrazone) tetrahydrochloride (Compound 42) as thetetrahydrate, mp 258°-9° C.

Compound 43, FIG. 7J.43: N,N'-bis(3,5-diacetylphenyl)octanediamide(0.100 g), aminoguanidine hydrochloride (0.115 g), and aminoguanidinedihydrochloride (10 mg) were heated in 95% ethanol (2.5 mL) for 20 hr.Cooling and filtration gave 0.17 g ofN,N'-bis(3,5-diacetylphenyl)octanediamide tetrakis(amidinohydrazone)tetrahydrochloride (Compound 43) as the 2.5-hydrate, mp 308°-9° C.

7. EXAMPLE: WHOLE CELL INHIBITION ASSAYS FOR UREA AND NO OUTPUT

This section describes in detail the methods and the results of a tissueculture assay to determine the ability of the compounds of the inventionto inhibit urea synthesis in activated macrophages.

7.1. MATERIAL AND METHODS

RAW 264.7 cells are plated in microculture wells at 1×10⁶ /ml in RPMI1640 with 10% FBS and otherwise standard culture conditions and allowedto adhere for 5 hours. They are then activated with 25 U/ml γ-IFN and0.1 μg/ml LPS in the presence of test inhibitors. The urea concentrationpresent in the supernatant media, after 18 hours of culture, isdetermined by a colorimetric blood urea nitrogen diacetylmonoxime assayperformed on an aliquot of the culture supernatant (Sigma Chem. Co., St.Louis, Mo.) and inhibition is expressed as the percentage urea comparedto that of a parallel control culture treated identically except thatthe concentration of added inhibitor is zero.

The production of nitrite is determined by assay of the same tissueculture supernatant by means of a colorimetric assay. Briefly, 4 partsof test solution containing 1% (w/v) sulfanilamide, 0.1%naphthylethylenediamine di-HCl (Griess Reagent), 2.5% H₃ PO₄ and onepart media are mixed, incubated for 10 minutes and the absorbance at 560nm determined. The nitrite concentrations are interpolated fromreference curves prepared using NaNO₂.

Various concentrations of the test compounds are included in the mediaof the stimulated RAW 264.7 cells in culture. The concentration of ureaand/or nitrite after 18 hours of activated culture is determined andcompared to the uninhibited control value obtained from at least oneparallel culture. The fractional reduction is calculated for eachconcentration of inhibitor and the IC₅₀ (concentration giving 50%reduction) is interpolated. Compounds were dissolved in media with mildheat (60° C.). Those with limited solubility were dissolved in base. Theconcentration of stock solutions of those compounds which were notcompletely soluble was determined by OD₂₈₀.

The culture supernatants are tested for the presence of an intracellularenzyme, lactate dehydrogenase (LDH) to determine the degree, if any, towhich the test compound has caused cell death. In the examples reportedhereinafter no such cellular toxicity due to the test compounds wasobserved.

7.2. RESULTS

The results of the examination of guanylhydrazone compounds to measuretheir capacity to inhibit urea production are presented in Tables I, IIand III. The most active compounds, reported in Table I, displayed IC₅₀of less than 10 μM. The most active compound was an tetraguanylhydrazonedecanediamide (Compound No. 14). Also highly active were thepentanediamide homolog of the above (Compound No. 13); the ethanedioxybis(guanylhydrazone), (Compound No. 16); a mixed ureabis(guanylhydrazone) (Compound No. 9); and triacetylbenzinetris(guanylhydrazone) (Compound No. 1).

Compounds having either one or two phenyl nuclei were effective. Whentwo nuclei were present their linkage by means of a urea, diamide oralkanedioxy functionality was effective. Test compounds having a singleguanylhydrazone functionality were much less effective.

                                      TABLE I                                     __________________________________________________________________________    RESULTS OF IN VITRO ASSAYS FOR COMPOUNDS                                      DEMONSTRATING HIGH LEVELS OF ACTIVITY                                                                              arginine                                                          urea  no.sub.2 /no.sub.3                                                                  transport                                                         %     %     %                                                           dose  suppression                                                                         suppression                                                                         suppression                                    # of         evaluated                                                                           of    of    of                                       Compound                                                                            Guanidino                                                                           Dissolved                                                                            (I.C..sub.50 In                                                                     induced                                                                             induced                                                                             induced                                  #     Groups                                                                              in     parenthesis)                                                                        production                                                                          production                                                                          peak                                     __________________________________________________________________________     1    3     5 mM   25 μM                                                                            100   72    40                                                   solution in                                                                          10 μM                                                                             60   58    20                                                   5 mM NaOH;                                                                           1  μM           0                                                   precipitation                                                                        (10                                                                              μM)                                                              when diluted                                                                  into RPMI                                                          9    3     5 mM   25 μM                                                                            100   71    68                                                   solution in                                                                          10 μM                                                                             60   53    32                                                   5 mM NaOH;                                                                           1  μM                                                                             0     0    14                                                   precipitation                                                                        (10                                                                              μM)                                                              when diluted                                                                  into RPMI                                                         13    4     5 mM   500                                                                              μM                                                                            100   52    93                                                   solution in                                                                          25 μM                                                                            100   43    19                                                   5 mM NaOH;                                                                           50 μM                                                                             87          7                                                   precipitation                                                                        25 μM                                                                             77                                                              when diluted                                                                         10 μM                                                                             45                                                              into RPMI                                                                            (10                                                                              μM)                                                  14    4     5 mM   200                                                                              μM                                                                            100   100   100                                                  solution in                                                                          50 μM                                                                            100   100    86                                                  5 mM NaOH,                                                                           10 μM                                                                             78    69    19                                                  forms a kind                                                                         2  μM                                                                             68    0     9                                                   of clot when                                                                         (1 μM)                                                              cold,                                                                         precipitation                                                                 when diluted                                                                  into RPMI                                                         15    2     Not soluble;                                                                         150                                                                              μM                                                                            100   100   100                                                  treat  15.0                                                                             μM                                                                            100    9     35                                                  as #11, 12, 15                                                                       5.0                                                                              μM                                                                             8     0     10                                                         (10                                                                              μM)                                                  16    2     Not soluble;                                                                         250                                                                              μM                                                                            100   100   100                                                  treat  25 μM                                                                            100    90    44                                                  as #11, 12, 15                                                                       5  μM                                                                             40    17    26                                                         (1-10                                                                            μM)                                                  __________________________________________________________________________

                                      TABLE II                                    __________________________________________________________________________    RESULTS OF IN VITRO ASSAYS FOR ACTIVE COMPOUNDS                                                                 UREA      NO.sub.2 /NO.sub.3                        # OF             DOSE TESTED                                                                            % SUPPRESSION                                                                           % SUPPRESSION                     COMPOUND                                                                              GUANIDINO                                                                             DISSOLVED                                                                              (I.C..sub.50 IN                                                                        OF INDUCED                                                                              OF INDUCED                        #       GROUPS  IN       PARENTHESIS)                                                                           PRODUCTION                                                                              PRODUCTION                        __________________________________________________________________________    11      2       Not soluble;                                                                           163  μM                                                                             100       100                                               dose     16.3 μM                                                                              0         0                                                defined by OD                                                                          5.4  μM                                                                              0         0                                                determination                                                                          (100 μM)                                                          (280 mM) of                                                                   SN of 5 mM                                                                    NaOH                                                                          solution +                                                                    heat + spin the                                                               suspension used                                                               as St. curve the                                                              ODs of #1, 9, 13                                              12      2       Not souluble,                                                                          100  μM                                                                             18-32      20                                               dose defined by                                                               OD determin-                                                                           (250 μM)?                                                         ation (at 280                                                                 μM) using as St.                                                           curve the ODs of                                                              #1, 9, 13                                                     17      2       Not soluble                                                                            177  μM                                                                              54-100   71-87                                             treated as                                                                    #11, 12, 15, 16                                                                        (150 μM)                                          18      2       Not soluble                                                                            233  μM                                                                             100       100                                               treated as                                                                             23.3      30        13                                               #11, 12, 15, 16                                                                        7.7       9         0                                                and 17   (25  μM)                                          19      2       Well dissolved                                                                         1    mM  100                                                         in PBS   500  μM                                                                             100                                                                  250  μM                                                                             100                                                                  100  μM                                                                              50                                                                  50   μM                                                                              30                                                                  10   μM                                                                              0                                                                   (100 μM)                                          20      2       5 mM solution in                                                                       400  μM                                                                             100        84                                               5 mM NaOH +                                                                            200  μM                                                                              38        15                                               heat     50   μM                                                                              19        0                                                         10   μM                                                                              16        0                                                         (300 μM)                                          __________________________________________________________________________

                                      TABLE III                                   __________________________________________________________________________    RESULTS OF IN VITRO UREA PRODUCTION ASSAYS                                    FOR COMPOUNDS DEMONSTRATING ACTIVITY                                                                             UREA                                               # OF              DOSE TESTED                                                                            % SUPPRESSION                              COMPOUND                                                                              GUANIDINO                                                                             DISSOLVED (I.C..sub.50 IN                                                                        OF INDUCED                                 #       GROUPS  IN        PARENTHESIS)                                                                           PRODUCTION                                 __________________________________________________________________________    2       2       Heat (60° C.-2 hrs)                                                              200  μM                                                                             100                                                        200 mM solution                                                                         20   μM                                                                             0-17                                                       in RPMI   (50  μM)                                         3       2       Heat (60° C.-2 hrs)                                                              100  μM                                                                             100                                                        1 mM solution in                                                                        50   μM                                                                             42                                                         RPMI      40   μM                                                                             21                                                                   1    μM                                                                              9                                                                   (500 μM)                                         4       1       Heat (60° C.-2 hrs)                                                              400  μM                                                                             100                                                        400 μM solution                                                                      200  μM                                                                              0                                                         in RPMI   100  μM                                                                              0                                                                   50   μM                                                                              0                                                                   20   μM                                                                              0                                                                   (300 μM)                                         5       2       Well dissolved                                                                          1    mM  73-100                                                     in PBS; 5 mM                                                                            500  μM                                                                             64                                                         solution  250  μM                                                                             52                                                                   125  μM                                                                             48                                                                   60   μM                                                                             37                                                                   30   μM                                                                             24                                                                   (250 μM)                                         6       1       Heat (60° C.-2 hrs)                                                              1    mM  100                                                        1 mM solution in                                                                        100  μM                                                                             40                                                         RPMI      50   μM                                                                              0                                                                   (250 μM)                                         7       1       Heat (60° C.-2 hrs)                                                              10   mM  100                                                        10 mM solution                                                                          2    mM  55                                                         in RPMI   1    mM  40                                                                   100  μM                                                                             35                                                                   10   μM                                                                             23                                                                   (2   mM)                                            8       2       Heat (60° C.-2 hrs)                                                              500  μM                                                                             100                                                        5 mM solution                                                                           50   μM                                                                             76                                                         in RPMI   (50  μM)                                         10      1       Heat (60° C.-2 hrs)                                                              400  μM                                                                              0                                                         400 μM solution                                                                      200  μM                                                                              0                                                                   20   μM                                                                              0                                                                   (No effect)                                         __________________________________________________________________________

8. EXAMPLE: WHOLE CELL INHIBITION ASSAY FOR ARGININE UPTAKE

This section describes in detail the methods and the results of a tissueculture assay to determine the ability of the compounds of the inventionto inhibit the uptake of arginine by activated macrophages.

8.1. MATERIALS AND METHODS

RAW 264.7 cells are plated in standard 96-well microculture plates at aconcentration of 10⁵ /well and allowed to adhere for from 2 hours toovernight. The medium is then replaced with medium containing thecompound to be tested. One hour later, the medium is supplemented so asto contain 25 U/ml γIFN and 0.1 μg/ml LPS and the cultures incubated afurther. The cells are then rinsed twice in HEPES- buffered Krebs saltsolution with 0.1% glucose. Carrier-free tetra-³ H-arginine is added toeach well (2.5 μCi/well with specific activity of 69 mCi/mmol) and theactive uptake of arginine is allowed to continue for a 5 minute periodafter which the cells are rapidly cooled to 0° C. by lavage with icedsaline containing 10 mM unlabeled arginine to displace any externallybound label. The contents of the washed cells are solubilized in 100 μlof formic acid and counted by standard techniques. The amount of tetra-³H-arginine uptake as a function of time incubation time is shown in FIG.6. In subsequent experiments to determine the activity of theinhibitors, incubations were performed for 8 hours.

8.2. RESULTS

The compounds of the present invention which were most active insuppressing urea production in activated macrophages were tested todetermine their effects on the uptake of arginine. The results, shown inTable I of Section 7.2, indicate that each of the active compounds wereeffective inhibitors of uptake at a dose similar to that whicheffectively inhibited urea production. These data suggest that anarginine transport protein is a target of action of these compounds.

9. EXAMPLE: ARGINASE INHIBITION ASSAY

This section describes in detail the methods and the results of acell-free assay to determine the ability of the compounds of theinvention to inhibit the activity of argininase obtained frommacrophages.

9.1. MATERIALS AND METHODS

A 1200 g×2 minutes supernatant is obtained from a cell lysate of washedRAW 264.7 cells made by the addition of a lysis buffer containing 50 mMHepes, 1% NP-40, 0.1 mg/ml phenylmethylsulfonylfluoride (PMSF), andaprotinin at 1 μg/ml. The volume of supernatant is adjusted so that theprotein concentration is between 2 and 4 mg/ml. A 1:4 mixture of thesupernatant in activation buffer (30 mM MnCl₂, 0.3M glycine, 1% BSA, pH9.8) containing various concentrations of the test inhibitor isincubated at 55° C. for 20 minutes. Arginase activity is determined bymixing a 1:2 the above solution and 0.375M arginine, pH 9.8 for 10minutes at 37° C. The urea concentration after this incubation isdetermined as above.

9.2. RESULTS

The six compounds most active in the suppression of cellular ureaproduction were tested to determine whether any could inhibit theactivity of arginase in the above-described cell lysate assay. Theresults do not indicate arginase to be sensitive to inhibition by thecompounds of the present invention at the concentrations and under theconditions employed.

10. EXAMPLE: TREATMENT OF CACHEXIA IN VIVO

An animal model of tumor-associated protein catabolic illness (cachexia)was employed to directly test the efficacy of Compound No. 14 inreducing whole-body nitrogen losses. Tumors were induced in theappropriate groups by intramuscular inoculation with 15×10⁶ AtT-20cells; this model is known to cause a protein catabolic illnesscharacterized by the consumption of normal quantities of food, but 20%net whole-body losses of protein within 14 days. Compound No. 14 wasadministered to the "treated" groups in a daily dose of 0.4 mg/Kg,intraperitoneal. Nude mice (nu/nu) were housed in metabolic cages (4 percage) to facilitate collection of daily urine samples for 14 days. Foodintake was measured daily. There were four groups of animals: 1)untreated, non-tumor bearing controls; 2) untreated, tumor-bearing; 3)treated, tumor-bearing, and 4) treated, non-tumor-bearing. Urine wascollected and urinary urea losses quantified.

The results are summarized in Table IV (expressed as mg urea/group):

                  TABLE IV                                                        ______________________________________                                        IN VIVO ACTIVITY OF COMPOUND #14 IN                                           SUPPRESSING WHOLE-BODY NITROGEN LOSS                                          URINARY UREA LOSS (mg/group)                                                  Day After          Tumor,    Tumor,  Controls,                                Tumor    Controls  Untreated Treated Treated                                  ______________________________________                                         2       189       240       195     193                                       4       252       342       368     285                                       6       256       508       295     397                                       7       302       456       312     247                                       8       287       300       275     257                                      10       177       259       206     216                                      11       240       279       219     239                                      12       230       230       115     203                                      13       192       186       201     242                                      14       161       238       187     157                                      Sum totals                                                                             2286      3038      2373    2436                                     ______________________________________                                    

The groups consumed similar quantities of food (16±2 g/day) during thisexperiment.

The data demonstrate that Compound No. 14 prevented excess urinary ureaexcretion normally associated with cachexia.

Since urea loss represents 80% of whole-body nitrogen losses, and thenitrogen intakes were similar in all groups, these data further suggestthat Compound No. 14 augments whole-body nitrogen retention.

The significance of these observations is demonstrated by estimating theimpact of these nitrogen losses to muscle mass. Compound No. 14prevented the loss of approximately 200 mg urea/mouse over the 14 dayperiod. This is equivalent to approximately 100 mg of nitrogen, 625 mgof protein, or 2.7 g of wet muscle mass that is retained by treatmentwith Compound No. 14.

11. EXAMPLE: TREATMENT OF INFLAMMATION IN VIVO

A standard animal model of inflammation was utilized to directly testthe efficacy of Compound No. 14 as an anti-inflammatory. Paw swellinginduced by injection of the irritant carrageenan into murine foot padshas been used to detect clinically useful anti-inflammatory drugs sincethe early 1960's.

Paw edema was induced by injection of 50 microliters of 1%Lambda-carrageenan in HEPES 25 mM, pH 7.4, into the planter surface ofthe left hindpaw of C3H/HeN mice, the right paw was injected with 50microliters of HEPES alone. The animals were divided into two groups:controls (n=3) received vehicle only, i.p. 1.5 hour before pawinjection; the experimental group (n=4) was treated with Compound No.14, 5 mg/Kg, i.p. 1.5 hour before paw injection. Three hours after pawinjection, paw thickness was measured using a caliper, and the dataexpressed as change of carrageenan paw vs HEPES paw thickness.

The results are summarized in Table V which contains thecarrageenan-induced increase in paw thickness in mm of three control andthree Compound No. 14 treated mice. The inhibition can also be reportedas percent inhibition of swelling as defined by the standard formula:Percent inhibition=(1-(treated/control))×100. Calculation of thisparameter for treated animals in which Compound No. 14 suppressedswelling yields a 70% inhibition.

                  TABLE V                                                         ______________________________________                                        EFFECT OF COMPOUND NO. 14 ON INFLAMMATION                                     CHANGE IN PAW THICKNESS                                                       Untreated     Treated With                                                    Controls      Compound #14                                                    ______________________________________                                        1.47          0.54                                                            1.50          0.41                                                            1.24          0.4                                                             ______________________________________                                    

FIG. 8 presents the effects of various doses of Compound No. 14 in thesame assay. The data show that doses of between about 1 and 10 mg/Kg ofbody weight are effective at inhibiting paw swelling.

These data demonstrate that Compound No. 14 prevented inflammation,which is believed to be mediated by the inhibition of arginine-transportdependent nitric oxide production in inflammatory cells.

12. EXAMPLE: COMPOUND NO. 14 HAS NO EFFECTS ON ENDOTHELIAL DERIVEDRELAXING FACTOR MEDIATED VASODILATORY RESPONSES

An important requirement of drug used to control the vascular collapseand hypostension caused by macrophage NO production is that it notinterfere with the activity of EDRF in vivo. Such interference can causean uncontrolled hypertension and has prevented the effective clinicaluse of NO-synthase inhibitors. We measured the effect of Compound No. 14on EDRF activity in an animal model and found that compound 14 inhibitsNO production yet does not inhibit EDRF activity.

Female Sprague-Dawley rats (225-250 g body weight) were anesthetizedwith nembutal (50 mg/Kg, i.p.), a tracheostomy tube was inserted, andthe carotid artery and jugular vein cannulated by standard methods usingpolyethylene tubing (PE 50). Tracey, K. J., et al., 1986, SCIENCE 234,470-474. Blood pressure was recorded continuously with a pressuretransducer and recorder (Model RS-3200, Gould Inc.,). In the experimentshown here, animals received a single sterile intra-arterial dose ofeither N^(G) -methyl-L-arginine (NMA; Sigma; 10 mg/Kg), Compound No. 14(50 mg/Kg), or vehicle (0.4 ml). Acetylcholine (ACh) diluted in LPS-freesterile water was administered via the jugular vein cannula at the dosesindicated. The solutions were diluted to produce a constant injectablevolume of 1 ml/Kg body weight.

The hypotensive (EDRF) response was measured as the decline in meanarterial blood pressure recorded 30 sec after administration of ACh. Thenumber of animals studied at each dose of acetylcholine was 4-6 for eachexperimental condition; data are expressed as the mean ±s.e.m.

EDRF activity was inhibited by NMA, evidenced by attenuated bloodpressure responses as compared to vehicle-treated controls (FIG. 9; seealso, Kilbourn, R. G., et al., 1990, PROC NATL. ACAD SCI. 87,3629-3632). In contrast, Compound No. 14 did not suppressacetylcholine-induced EDRF activity in vivo, indicating that CompoundNo. 14-treated animals retained the functional capacity forendothelial-derived NO activity.

13. EXAMPLE: COMPOUND NO. 14 PREVENTS FATAL ENDOTOXIC SHOCK

Experiments were undertaken to evaluate the effects of Compound No. 14in preventing the lethal toxicity of lipopolysaccharide (LPS). LPS wasadministered to induce 50% lethality within 72 hr in BALB/c mice (FIG.10). BALB/c mice (19-21 g) were given LPS (E. coli 0111:B4; Sigma) in adose of 13.75 mg/Kg by intraperitoneal injection (0.2 ml/mouse). StockLPS solutions (10 mg/ml) were sonicated initially for 20 min, diluted inLPS-free water (1.375 mg/ml), then sonicated again for 10 minimmediately prior to injection. Compound No. 14 (1 mg/Kg, i.p.) wasadministered 1.5 hr before LPS. Compound No. 14 injectate was free ofLPS as measured by quantitative chromogenic Limulus amebocyte lysatetest (BioWhittaker, Walkersville, Md.). Data points are from two groupsconsisting of 10 mice per group.

Compound No. 14 administered 1.5 hours before LPS reduced lethality.Data were subjected to statistical analysis using the z test forindependent proportions. The difference bewteen control and Compound No.14 is significant (one-tailed p value <0.05; z=1.95).

There were no gross signs of systemic toxicity in animals receivingCompound No. 14 alone. After LPS, controls were ill-kempt, had decreasedmobility, and huddled together. These visible signs of LPS toxicity weremarkedly suppressed by Compound No. 14. Diarrhea occurred in allanimals, and was not suppressed by Compound No. 14.

Previously available NOS inhibitors have had limited success inimproving survival from endotoxemia, in part because theyindiscriminately suppress EDRF. Cobb, J. P., 1992, J. EXP. MED.176:1175-1182; Minnard, E. A., 1994, ARCH SURG. 129:142-148; Billiar, T.R., 1990, J. LEUKOCYTE. BIOL. 48:565-569. Suppression of EDRF duringendotoxemia may impair survival by causing vasoconstriction and adiminution of blood flow to critical vascular beds. The present data nowindicate that inhibiting cytokine-inducible macrophage NO with an agentthat preserves endothelial-derived NO responses can confer a survivaladvantage during septic shock.

14. EXAMPLE: COMPOUND NO. 14 PREVENTS THE PRODUCTION OF CYTOKINES AND NO

This section describes in detail the methods and the results of a tissueculture assay to determine the ability of the compounds of the inventionto inhibit the production of TNF by activated macrophages, and shows,for purposes of explanation and not limitation, that Compound No. 14 iseffective in blocking the secretion of TNF by a mechanism that isindependent of the inhibition of arginine uptake.

RAW 264.7 cells were plated in standard 6-well culture plates at adensity of 10⁶ /well and allowed to adhere for from 2 hr to overnight.The medium was then replaced with medium containing the compound to betested. One hour later, the medium was supplemented so as to contain 25U/ml γIFN and 0.1 μg/ml LPS and the cultures incubated a further 18 hr.The medium was then collected, and cell debris removed bycentrifugation. This conditioned supernatant was then assayed for thepresence of TNF using standard methodologies: L929 cell cytotoxicitybioassay, radioimmunoassay or ELISA, and Western blotting withantibodies against murine TNF.

FIG. 11 shows that in the presence of increasing amounts of Compound No.14, the production of bioactive TNF as determined by L929 cell bioassayby RAW 264.7 cells was prevented. The data show that at 10 μM CompoundNo. 14 there was a reduction of more than 99% of the TNF accumulation inthe culture medium. FIG. 12 shows a Western blot that demonstrates theabsence of TNF protein in the medium of cells cultured with 5 and 25 μMconcentrations of Compound No. 14. The failure of Compound No.14-treated cells to secrete TNF can not be simply attributed to theinhibition of NO synthesis by Compound No. 14. The NOS inhibitorN-methyl-arginine (NMA), even at concentrations of 10 μM, did notinhibit the production of TNF in this system. The inhibition of TNFproduction was also not attributable to the effects of argininedepletion brought on by the blockage of arginine transport. FIG. 13Ashows that Compound No. 14, at a 5 μM concentration, functioned as aninhibitor of NO synthesis, but that this inhibition could be partiallyovercome by the presence of between about 50 and 100 μM arginine in themedium. By contrast the data in FIG. 13B clearly show that the effectson TNF secretion of Compound No. 14 at 5 μM were not reduced even by asmuch as 1.0 mM extracellular arginine. These results show that CompoundNo. 14 specifically blocked the production of TNF from activatedmacrophages by a mechanism that does not critically depend on arginine.

Qualitatively similar data has been obtained by measurement of the serumTNF levels in rats stimulated to produce TNF by parenteral LPSadministration. Rats were given Compound No. 14, i.p., 2.0 hours priorto LPS stimulation. The serum levels of TNF were measured at between 2and 3 hours after LPS stimulation. Compound No. 14-treated animalsshowed only about half the level of serum TNF as found in the untreatedcontrols.

Similar results concerning a variety of cytokines in addition to TNF,e.g., IL-6 and Macrophage Inflammatory Proteins-1α and -1β (MIP-1α andMIP-1β) were obtained using human peripheral blood as a source ofmonocytes. Human peripheral blood mononuclear cells (PBMC) were isolatedusing "FICOLL®"-based methods and plated in standard 6-well cultureplates at a concentration of 10⁶ /well and allowed to adhere for from 2hr to overnight whereupon non-adherent cells were washed out and themedium was then replaced with medium containing the compound to betested. One hour later, the medium was supplemented so as to contain 25U/ml γIFN and 0.1 μg/ml LPS and the cultures incubated a further 18 hr.The medium was then collected, and cell debris removed bycentrifugation. This conditioned supernatant was then assayed for thepresence of cytokines using the standard methodologies of L929 cellcytotoxicity bioassay and immunoassay (ELISA). The results indicatedthat, when tested in this system, Compound No. 14 effectively inhibitedthe production of TNF, IL-6, MIP-1α, and MIP-1β by human PBMC cells at aconcentration of about 10-20 μM. FIGS. 14A-D show that in the presenceof increasing amounts of Compound No. 14 from 1 to 20 μM, the productionof these cytokines by human PBMC cell cultures was prevented.

15. EXAMPLE: COMPOUND NO. 14 CONFERS PROTECTION FROM FOCAL CEREBRALINFARCTION

This section describes in detail the methods and the results of ananimal model to determine the ability of Compound No. 14 to treatcerebral infarction (also known as brain infarction or "stroke").

Lewis rats (male, 270-300 g) were given food and water ad libitum beforeand after surgery. Animals were anesthetized with ketamine (120 mg/Kgi.m.), allowed to breathe spontaneously, and body temperature maintainedat 35.5°-36.5° C. with a heating blanket. The ventral neck and areabetween the right eye and ear was shaved. A midline ventral cervicalincision was used to expose the left common carotid artery (CCA) whichwas dissected free from surrounding tissue with preservation of thevagus nerve. A loop of 4-0 silk was then placed around the artery forfuture manipulation. The right common carotid artery was then exposedand permanently occluded with double 4-0 silk ligatures.

To perform the craniotomy, a 1 cm incision was made orthogonal to theline joining the external auditory canal and the lateral canthus of theright eye. With the aid of a dissecting microscope, the right middlecerebral artery was exposed through a 1 mm burr hole drilledapproximately 2 cm rostral to the fusion of the zygoma with the temporalbone. Drilling was performed under a continuous drip of normal saline toavoid transmission of heat to the underlying cortex. The bone wasthinned with the drill, leaving a thin shell which was removed with amicro-hook and micro-forceps. The dura mater was then cut and reflectedwith a 30 gauge needle, exposing the right middle cerebral artery (MCA)approximately 1 mm from the rhinal fissure.

Using a micromanipulator and a 20 micron tungsten wire hook, the rightmiddle cerebral artery was elevated 0.5 mm above the cortical surfaceand divided by application of an electrocautery tip to the tungsten hookabove the vessel. The application of heat quickly cauterized and severedthe artery which fell back onto the cortex with no underlying corticalinjury. To cause a reproducible stroke the left CCA must be temporarilyoccluded for 30-60 minutes. Accordingly, in the present model the leftCCA was occluded for 30 minutes. Surgical gelfoam was placed over thecraniotomy defect, and the skin incisions closed with a vicryl sutures.The animals were then returned to their cages, where they were allowedfree access to food and water for 24 hours. After surgery, animals weresomewhat clumsy but resumed activities including walking, eating, anddrinking.

Twenty-four hours after MCA severing, animals were anesthetized anddecapitated and the brains were quickly removed without perfusion andcoronally sectioned at 1 mm intervals with a brain slicer for analysis.Freshly prepared slices were immersed and incubated in2,3,5-triphenyltetrazolium (TTC) (2% in NaCl, 154 mM) for 30 minutes at37° C. in the dark to stain for mitochondrial dehydrogenase activity.Brain infarctions were visualized as areas of unstained (white) tissuewhich were easily contrasted with viable tissue which stained red.Slices were then placed in buffered 10% formalin and infarct areadetermined by planimetry on projected images of photographed brainslices. Infarct size for an individual animal was calculated by summingthe infarct area present on each brain slice and dividing by the sum ofhemisphere area for all slices for that animal (expressed as apercentage of hemisphere area for each slice). Animals were studied ingroups of 10, and the data expressed as average stroke volume for thegroup.

The mean infarct size of controls (not treated with Compound No. 14) wasobserved to be 3.1%±0.5%. Animals that received Compound No. 14 (1mg/Kg, i.v.) one hour before the artery was severed developed smallerstroke size (1.7%±0.2%). These differences were statisticallysignificant (p<0.05) by Student's t-test for unpaired data. Theseexperiments indicated that Compound No. 14 effectively reduced the sizeof focal cerebral infarction.

16. EXAMPLE: ANTI-NEOPLASTIC ACTIVITY OF COMPOUND NO. 14

This section describes in detail the methods and results of an animalmodel of tumor growth to determine the ability of the compounds of theinvention to inhibit tumor growth, and cause regression of tumors. Cellsare plated in standard tissue culture flasks in DMEM supplemented withfetal calf serum (10%). Chinese hamster ovary (CHO) cells stablytransfected with a mammalian expression vector constitutively secretehuman TNF (CHO-TNF). The experiments with the CHO-TNF tumor wereperformed as follows: on the day of injection into nude mice, cells areharvested and injected intramuscularly (10-15×10⁶ cells per animal) intothe hindlimb of nu/nu nude mice (20 g body weight). Animals are housedand provided food and water ad libitum. Tumor growth is monitored, andwhen established tumors are present (during week six) the test compoundsare administered daily (0.4 mg/Kg intraperitoneal, once daily). Aftertwo weeks the animals are euthanized, the tumor weighed, and measuredwith a caliper. The CHO-TNF tumor also metastasizes to the skin of theseanimals; the number of metastases is scored.

Parenteral administration of Compound No. 14 caused a reduction in tumorsize in four out of five animals. The tumors of these animals wereweighed and their size determined. Examination of the mice for skinmetastases revealed that an average of 2.5±1 skin metastases developedin 4 out of 5 controls; skin metastases were not present in any of thetreated animals. These data show that Compound No. 14 has anti-tumoractivity.

    ______________________________________                                        TUMOR          TUMOR                                                          WEIGHT         DIMENSION    # of                                              (g)            (mm.sup.2)   METASTASES                                        ______________________________________                                        CON-    2.686 ± 0.77                                                                          343.58 ± 64.1                                                                           2.5 ± 1.1                                  TROL                                                                          (n = 5)                                                                       No. 14  1.628 ± 1.07                                                                          207.99 ± 119.90                                                                         0                                             (n = 4)                                                                       ______________________________________                                    

17. INHIBITORY EFFECTS OF COMPOUND NO. 14 ON ARGININE UPTAKE AND NOOUTPUT OF PREVIOUSLY QUIESCENT VERSUS ACTIVATED CELLS

This section describes experiments that measure the concentration ofCompound No. 14 needed to inhibit arginine uptake and NO output. Thetechniques employed were described in section 7.1 supra.

FIG. 13A shows the effects of Compound No. 14 on the output of NO by RAW264.7 cells that have been continuously exposed to Compound No. 14 fromone hour prior to γ-IFN/LPS stimulation until completion of the assay 18hours after stimulation. The data show that Compound No. 14 was ainhibitor of NO output that was competitively antagonized byextracellular arginine. These data indicate that the I.C.₅₀ for CompoundNo. 14 in this assay, at physiologic arginine concentrations (100 μM),was between 3 and 5 μM.

As shown in FIG. 6, the peak level of arginine uptake by RAW 264.7 cellsoccured at about 8 hours after γ-IFN/LPS stimulation. When RAW 264.7cells were exposed to Compound No. 14 prior to stimulation, as in FIG.13B, and the level of arginine uptake measured at 8 hours, the I.C.₅₀for arginine uptake of Compound No. 14 was very similar that observedfor NO output, 7.5 μM (data not shown).

All effects of Compound No. 14 on macrophage NO output, however, couldnot be entirely attributed to an acute blockade of arginine transport.FIG. 15 shows the results observed when RAW 264.7 cells wereγ-IFN/LPS-stimulated in the absence of any inhibitor and, 8 hoursthereafter, the cells were exposed for 10 minutes to Compound No. 14,whereupon arginine uptake was measured. An I.C.₅₀ =60±15 μM (mean±std.err., n=3) was measured when RAW 264.7 cells, that had beenγ-IFN/LPS-stimulated 8 hours previously were exposed to Compound No. 14in arginine-free buffer for 10 minutes prior to assay of arginineuptake. The results of these acute exposure experiments differed fromthe previously discussed, continuous exposure results in two ways.Firstly, approximately one third of the total arginine uptake occuredthrough a Compound No. 14-independent mechanism which appeared to bepresent in unstimulated as well as stimulated RAW 264.7 cells. Secondly,the concentration of Compound No. 14 needed to 50% inhibit arginineuptake after γ-IFN/LPS-induced arginine transport has been establishedfor a period of hours was greater than that needed to prevent 50% of theincreased arginine uptake or NO output when Compound No. 14 wasintroduced prior to stimulation.

An intermediate level of sensitivity to Compound No. 14 was observedwhen γ-IFN/LPS-stimulated RAW 264.7 cells were exposed to inhibitor from8 hours after the initial stimulation until the end of the experiment.In these experiments, NO output was measured during hours 12-24. FIG. 16demonstrates that under such experimental conditions an I.C.₅₀ of 20±2μM (mean±std. err., n=3) was observed.

Together these data show that when RAW 264.7 cells were exposed toCompound No. 14 prior to activation by γ-IFN/LPS, NO output andinducible arginine uptake were equally sensitive to low levels ofCompound No. 14, while the arginine uptake of unstimulated PAW 264.7 wasinsensitive to Compound No. 14. By contrast, the arginine uptake of RAW264.7 cells, stimulated with γ-IFN/LPS, and exposed briefly to CompoundNo. 14 after a delay was less sensitive to of Compound No. 14 than whenCompound No. 14 was given prior to stimulation. Likewise the NO outputof cells treated with Compound No. 14 hours after stimulation was shownto be less sensitive to inhibition by Compound No. 14 than was theinduction of NO production by previously quiescent cells.

These data indicate that higher levels of Compound No. 14 will berequired, in vivo, to block the ongoing production of NO by activatedmacrophages than would be needed to prevent the initiation of NOproduction by non-activated macrophages and that Compound No. 14 will bemore effective when chronically applied than when acutely applied tocells.

The present invention is not to be limited in scope by the specificembodiments described which were intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components were within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

What is claimed is:
 1. A compound having the formula: ##STR5## wherein:X₂ =GhyCH--, GhyCCH₃ -- or H--;X₁, X'₁ and X'₂, independently =GhyCH--or GhyCCH₃ --; --Z=--A--(CH₂)_(n) --A--, n=2-10, and A=--NH(CO)--, andsalts thereof.
 2. The compound of claim 1 wherein, when X₂ is GhyCH-- orGhyCCH₃ --, X₂ is meta or para to X₁ and X'₂ is meta or para to X'₁. 3.The compound N,N'-bis(3,5-diacetylphenyl)decanediamidetetrakis(amidinohydrazone) tetrahydrochloride.
 4. The pharmaceuticalcomposition comprising the compound of claim 1 and a pharmaceuticallyacceptable carrier.
 5. The pharmaceutical composition comprising thecompound of claim 2 and a pharmaceutically acceptable carrier.
 6. Thepharmaceutical composition comprising the compound of claim 3 and apharmaceutically acceptable carrier.